310 10 * 1mm Stainless simbi yakasungirirwa tubing kemikari chikamu, Iyo N-terminal domains yespidroin inoumba mahydrogels anobva pane amyloid fibrils uye inopa chikuva cheprotein immobilization.

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310 10 * 1mm Stainless simbi yakasungirirwa tubing vatengesi

Kuumbwa Kwemakemikari

Mechanical Properties

Recombinant spider silk mapuroteni (spider silk proteins) ane akawanda anogona kushandiswa mukuvandudza biomaterials mitsva, asi yavo multimodal uye aggregation-prone nature inoita kuti zviome kuwana uye nyore kushandisa.Pano tinoshuma kuti recombinant miniature spidroin mapuroteni uye, zvakakosha, iyo N-terminal domain (NT) pachayo inokurumidza kuumba anozvitsigira uye akajeka mahydrogel pa 37 ° C.fusion mapuroteni anosanganisira NT uye yegirini fluorescent protein kana purine nucleoside phosphorylase inoumba yakazara inoshanda fusion mapuroteni.Hydrogels.Mhedzisiro yedu inoratidza kuti recombinant NT uye mapuroteni efusion anopa goho rekutaura uye endow hydrogels ane zvinoyevedza zvimiro zvakaita sepachena, gelation pasina kuyambuka, uye yakananga immobilization yemapuroteni anoshanda pakunyanya density.
Madandemutande ane mapoka manomwe akasiyana ematehwe esirika, rimwe nerimwe richibudisa rudzi rwakati rwesirika.Ese marudzi manomwe esirika anoumbwa nemaprotein espider silk (spidroins) angangoita 6000 masara akareba uye ane hombe yepakati kudzokorora dunhu rakakomberedzwa nespherical N- uye C-terminal domains (NT uye CT)1,2.Rudzi rwakadzidzwa zvakanyanya rwesirika, iyo yekutanga ampulla, inogadzirwa neiyo primary ampulla gland.Mugland iyi, monolayer yemasero epithelial inogadzira mapuroteni espidroin uye anoaisa mulumen yegland, apo anenge aripo mune inonyunguduka fomu (doping) pane yakanyanya kuwanda (30-50% w/v)3,4.Kurongeka uye kuumbwa kwemapuroteni makuru e-ampullar spidroin mugland yakakakavadzana, asi humbowo huzhinji hwekuedza hunoratidza kuvapo kwekazhinji helical uye / kana random helical conformation uye micellar kana lamellar zvimiro5,6,7,8,9,10.Kunyange zvazvo nzvimbo dzinodzokororwa dzichigadzirisa maitiro emagetsi e-silk fibers, kugadzira β-sheet nanocrystals uye amorphous structures11,12,13,14,15, magumo ekupedzisira anodzora silk fibers maererano nekuchinja kwemamiriro ezvinhu pamwe chete nesiriki gland16,17,18.Nekudzora kuumbwa kwesirika, 19. Nzvimbo dzekupedzisira dzinochengetedzwa nekushanduka-shanduka uye basa ravo rinogona kunge rakajairika kune ese mapuroteni e spidroin 2,20,21.Munguva yekufamba nemukati, pH ye spidroin inoderera kubva pa7.6 kusvika ku <5.716 uye inowedzera neshear uye kutambanudza kunopindirana nekufamba kuburikidza nekufamba zvishoma nezvishoma.Mukugadzirisa, CT is an α-helical constitutive parallel dimer17, asi mukupindura pasi pH uye shear forces, CT inoputika uye inoshandura β-layers16, 17, zvichida inokonzera β-layers munzvimbo dzinodzokorora dzeShandura 16. NT ndeye monomeric pasi mamiriro anoratidza mamiriro emu lumen yegland uye anoyananisa kunyungudika kwe spidroin, asi pakuderedzwa pH, protonation yenhamba ye carboxylic acid side cheni inotungamira ku dimerization ye NT ine pKa inosvika 6.5, nokudaro kudzikamisa NT uye kugadzirisa spidroin muhombe. uwandu.network16,18.Nokudaro, NT inoita basa rakakosha mukugadzira filament, kushanduka kubva kumonoma mucoating kuenda ku dimer mu fiber23,24,25.NT inoramba yakanyungudika uye helical pasi pemamiriro ese akadzidzwa kusvika nhasi16, 18, 19, 20, 26, 27, 28, 29, iyo yakakurudzira kuvandudzwa kwayo seyekunyungudika-inosimudzira label yekugadzirwa kweheterologous proteins.
Recombinant mini spider silk protein, ine imwe NT, imwe pfupi yekudzokorora dunhu, imwe CT, uye His6 tag (His-NT2RepCT) yekucheneswa, inonyungudika mumvura inoyerera seprotein yespider silk uye inotevedzera hunhu hwakakosha hwedandemutande. .chinyorwa 25.31.His-NT2RepCT inogona kurukwa kuita faibha dzinoenderera uchishandisa muchina webiomimetic umo pH 8 yakanyungudika coating inobuditswa mupH 525,32,33,34,35 yekugezera yemvura.Kuviriswa kweBioreactor kweE. coli kuratidza Yake-NT2RepCT uye kurapwa kwakazotevera kwakakonzera > 14 g/L goho mushure mekucheneswa.Goho repamusoro, kunyungudika kwepamusoro, uye mhinduro yakakwana yeHis-NT2RepCT kune acidic mamiriro ese anonzi NT23, 25, 34.
Pano tinoshuma kukurumidza kuumbwa kwema hydrogels akajeka kubva kune recombinant spidroin mapuroteni, kusanganisira NT chete, nekupinza mhinduro yeprotein pa37 °C.Tichishandisa thioflavin T fluorescence (ThT), Fourier inoshandura infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR) uye transmission electron microscopy (TEM), takaona kuti NT nemamicrospider mapuroteni anochinja magadzirirwo kuita β-mashizha uye amyloid-like fibrils. apo gels inoumbwa.Mukuwedzera, mapuroteni ekubatanidza eNT uye green fluorescent protein (GFP) kana purine nucleoside phosphorylase (PNP) inoumba mahydrogel ane zvizere zvinoshanda zvakasanganiswa zvimedu.High-throughput expression in heterologous hosts, pamwe nekukurumidza kuumbwa kwema hydrogel pasi pemamiriro emuviri, inovhura mukana wekuita zvinodhura kugadzirwa kwemahydrogel ane mabasa einjiniya.
Kusiyana nevakawanda vanoshumwa recombinant spidroin proteins36, His-NT2RepCT yakagadzikana muTris-HCl buffer papH 8 uye inogona kuiswa kusvika ku500 mg/mL pasina kunaya kwemvura25.Nokudaro, takashamiswa kuona kuti puroteni iyi inokurumidza kuumba optical clear, self-supporting hydrogels painoputirwa pa 37 ° C (Fig. 1b-d).Zvimwe zvidzidzo zvakaratidza kuti His-NT2RepCT gelation yakaitika pamusoro pehuwandu hwakawanda hweprotein concentrations (10-300 mg / mL) uye kuti iyi fungidziro yakanga yakanyatsopindirana nenguva ye gelation (Fig. 1c uye Supplementary Fig. 1).Kuti tizive kuti ndezvipi zvikamu zveYake-NT2RepCT mediate hydrogel kuumbwa, isu takazoongorora imwe neimwe dura pachedu uye mumasanganiswa akasiyana tichishandisa flask inversion assay (Mufananidzo 1a, b).Zvose zvakaedzwa zvikamu zve recombinant spidroin zvakagadzira gels (paprotein concentration ye 300 mg / mL) musingasviki 1 h, kunze kweiyo precipitated 2Rep (Fig. 1b).Izvi zvinoratidza kuti NT uye CT chete, pamwe chete, kana kuti yakabatana nekudzokorora, inogona gel pa 37 ° C uye kuti His6 tag haikanganisi iyi nzira kune chero ipi zvayo inokosha.Tichifunga nezvepfungwa yakajairika yekuti NT ipuroteni inonyungudika uye yakagadzikana, uye kuti mishumo yapfuura yezve recombinant spidroin hydrogels yakati gelation mhedzisiro kune conformational shanduko mumatunhu anodzokororwa uye/kana maCTs, NT pachayo inogona.Kuwanikwa kwegelation kwaisatarisirwa.Supplementary Table 1) 37, 38, 39. Zvinoshamisa, NT yakatove gelled mukati memaminitsi e10 pamusangano we ≥ 300 mg / mL (Fig. 1c).Vial inversion kuyedza neakasiyana siyana etestamende itsva yakaratidza kuti pa> 50 mg/mL mhinduro yeNT yakakurumidza kupfuura Yake-NT2RepCT pachikamu chinoenderana (w/v, Mufananidzo 1c).
Schematic inomiririra yeakasiyana spidroin anovaka akadzidzwa mubasa iri.b Gel nguva pa 37 ° C yezvakasiyana-siyana recombinant spidroin proteins (300 mg/mL) inosimbiswa nekupidigura vial.CT gel pakarepo pasina incubation (<300 mg/mL), 2Rep precipitates (300 mg/mL, 5 mm chiyero).c Gel nguva yeHis-NT2RepCT uye NT pane yakaratidzwa maprotein concentrations pa37°C.d Mifananidzo yeHis-NT2RepCT uye NT mahydrogel ane dandemutande uye vara rekuti “NT” rakadhindwa pazasi, zvichiteerana (zvose 200 mg/mL, scale bar 5 mm).
Hydrogels inoumbwa neakasiyana-siyana recombinant spidroin mapuroteni ane mavara akasiyana zvishoma, uye ziso rakashama rakatarisa rinoratidza madhigirii akasiyana ekujeka (Fig. 1b).NT majirosi akajeka zvakasarudzika nepo mamwe majerori achive opaque.His-NT2RepCT uye NT gels akakandwa mucylindrical chubhu aigona kubviswa muforoma intact (Fig. 1d).
Kuti uedze kana spider silk coatings gel pasi pemamiriro ezvinhu zvino awanikwa achikonzera gelation yemapuroteni e-spidroin, machira akaunganidzwa kubva kune hombe ampulla gland yeSwedish bridge spider (Larinioides sclopetarius).Zvipfeko zvakachengetwa mu 20 mM Tris-HCl buffer pa 50 mg / mL (zvichienderana nekuyerwa kwakaoma uremu), asi hapana gelation yakaonekwa mukati mezuva re21 incubation pa 37 ° C (Supplementary Figure 2a).
Kuverengera aya gels, kuyerwa kwerheological kunogona kushandiswa kudzidza maitiro eiyo gelation uye kuona iyo yakazara mechanic zvimiro.Kunyanya, kutarisa modulus yekuchengetera (elasticity) pane yakakwirira tembiricha inogona kupa ruzivo nezve gelling tembiricha pamwe neiyo viscoelastic zvimiro zvejasi.Kuyedza kukwira kwekushisa (uchishandisa 1 ° C/min pa 25-45 ° C, zvichibva pane zvidzidzo zvekare uchishandisa zvigadziriswa silk stock solutions) 40,41 yakaratidza kuti moduli yekuchengetedza yeHi-NT2RepCT uye NT mhinduro yakawedzera nekuwedzera kwekushisa.yakawedzerwa (Fig. 2 uye Supplementary Fig. 3).Zvinokosha, iyo NT module yakatanga kukura patembiricha yakaderera kana ichienzaniswa neYake-NT2RepCT, inoenderana nekukurumidza gel nguva yakaonekwa apo NT yakanyatso nyuchirwa neHis-NT2RepCT pa37 ° C (Mufananidzo 1).Mushure mekudonha kwakatevera kwekushisa, iyo yekuchengetedza modulus haina kudzokera kune yakaderera tsika uye yakaramba iri pamusoro pekurasikirwa modulus (ona Supplementary Fig. 3), ichiratidza thermally isingachinjiki yakagadzikana gelation.Mushure mekuita gelation, iyo yekupedzisira elastic modulus yakabva pa15 kusvika 330 kPa yeHis-NT2RepCT hydrogels pamusangano we100-500 mg/mL, uye yekupedzisira elastic modulus yeNT hydrogels (100-500 mg/mL) kubva pa2 kusvika 1400. kPa (Fig., 2 uye yakazara ramp data) ona Supplementary Fig. 3).
a Shanduko yetembiricha panguva yekuyera His-NT2RepCT (300 mg/mL) uye b NT (300 mg/mL) nekudedera.Miseve inoratidza maitiro ekupisa, uye kupenya kwakareruka kwenzvimbo yekuchengetedza data kunoratidza kuyedzwa kune yakaderera torque kukosha kune chiridzwa pane yakatsanangurwa nemugadziri, chiri chikonzero chekuwedzera ruzha.c End-module kuunganidzwa kweHis-NT2RepCT uye NT mushure mekukwira kwekushisa (100, 300, uye 500 mg/mL).Kuverengera kwese kwemodule kunotorwa pamwero we 0.1 Hz.
Senzira inogona kuferefeta shanduko ye conformational ine chekuita negelation, takarekodha FTIR spectra yeHis-NT2RepCT uye NT isati yasvika uye mushure megelation pa37 ° C (Mufananidzo 3a,b).Sezvaitarisirwa, iyo spectra yeHi-NT2RepCT uye NT mhinduro dzakaenderana nemapuroteni anoratidza α-helix/random coil yechipiri chimiro, ine bhendi rakadudzwa pa1645 cm-1.Kune ese ma hydrogels, gelation yakakonzera kuumbwa kwemaoko maviri pakati I band paanenge 1617 cm-1 uye 1695 cm-1 (Fig. 3a, b), zvichiratidza kuumbwa kwe antiparallel β-sheet structures.Shanduko idzi dzinogonawo kuoneka zvakajeka mune inoteedzana yechipiri derivative uye musiyano gelation spectra (Supplementary Fig. 4b).Iwo mabhendi maviri eNT β-layer ainyanya kutaurwa kupfuura ayo eHis-NT2RepCT, zvichiratidza kuti zvese zviri mukati me β-layer bands muNT hydrogel yaive yakakwira kupfuura yeNT2RepCT hydrogel.
a FTIR absorption spectra yeHis-NT2RepCT uye b NT (zvose 500 mg/mL) isati (solution) uye mushure (gel) incubation pa37°C.c TEM mifananidzo yakamiswazve 50 mg/ml NT2RepCT gels uye d NT.Scale bar 200 nm.e Fiber madhayamita eHis-NT2RepCT uye NT mahydrogel.n = 100 akayera fibrils, p <0.0001.Mabhawa ekukanganisa anoratidza kutsauka kwakajairwa.Pakati pezvipingamupinyi mbariro ndiyo kureva.An unpaired t-test (two-tailed) yakashandiswa pakuongorora nhamba.f ThT fluorescence yeakasiyana recombinant spidroin proteins (100 mg/mL) pa 37 °C pasina kuzununguka.g NT (100 mg/mL) maekiseni ekuita jekiseni kubva pa100 mg/mL NT gel ine 0%, 5%, 10%, uye 20%.
Kuongororwa kwejeri kushandisa transmission electron microscopy (TEM) yakaratidza kuti hydrogel inosanganisira amyloid-like fibrils (Figs. 3c, 3d).NT-yakaumbwa fibrils yakarebeswa (5-12 nm muhupamhi) uye isina matavi, nepo His-NT2RepCT fibrils yaive mapfupi pakureba uye yakafara zvakanyanya mudhayamita (7-16 nm) (Fig. 3e).Mhedzisiro iyi yakatibvumira kutevera kinetics ye fibrosis tichishandisa thioflavin T (ThT) assay.Kune ese anodzokorora mapuroteni espidroin, chiratidzo chefluorescent chakawedzera apo sampuli dzakaiswa pa37 ° C (Fig. 3f, Supplementary Fig. 5a).Zvichienderana nekutsvaga uku, kuongororwa kwe microscopic ye NT uye His-NT2RepCT pasi pemamiriro ekugadzirisa kwakaratidza kuwedzera kweyunifomu muThT fluorescence pasina kuoneka kwekuunganidza kweThT-positive aggregates (Supplementary Fig. 5b, c).Kuumbwa kweThT-positive fibrils kwakanga kusina kufambidzana nekuwedzera kweNT uye Yake-NTCT turbidity (Supplementary Fig. 5d), izvo zvinoreva kuti network yefibrils mu gel inogona kuumba pasina kukanganisa kujekesa gel.Kudyara mbeu nekuwedzera zvidimbu zvidiki zvefibril zvakafanoumbwa zvinogona kukurumidzisa kuumbwa kwefibril mamwe maamyloids42,43,44 asi kuwedzera 5%, 10% kana 20% (w/w) NT kumushonga weNT hydrocoagulants.seeding effect (Fig. 3g).Zvichida izvi zvinokonzerwa nekuti iyo fibrils mu hydrogel yakanyatsogadziriswa uye haigoni kushandiswa sembeu.
Maitiro asingatarisirwe eanodzokorodza spidroin mapuroteni pakupisa kwakanyanya akakurudzira imwe nyukliya magnetic resonance (NMR) spectroscopy zvidzidzo kuona shanduko dzinoenderana nekuumbwa kwegel.NMR spectra yeHis-NT2RepCT mhinduro dzakarekodhwa nekufamba kwenguva pa37 ° C yakaratidza kuti CT yakanga ichiri kupetwa zvishoma, nepo NT uye 2Rep masaini akange anyangarika (Fig. 4a), zvichiratidza kuti yainyanya NT uye 2Rep iyo yakadzora kuumbwa kweYake- NT2RepCT hydrogel.Chiratidzo cheC CT chakaderedzwawo kusvika ku20% yehukuru hwayo hwepakutanga, zvichiratidza kuti CT inowanzogadziriswa uye inobatanidzwa mune hydrogel chimiro.Kune chidimbu chidiki cheCT, icho chinofamba semu preincubated sample uye nekudaro ichicherechedzwa nemhinduro NMR, iyo spectra inoshaya masaini kune gumi ekutanga akaumbwa asara, pamwe nekuda kwekuoma kwekusamira kwechikamu chakabatanidzwa cheHis-NT2Rep .Mucherechedzo weNMR we -state of hydrogels -NT2RepCT yakaratidza kuvapo kwakanyanya kwe-α-helices uye β-layers uye, kusvika zvishoma, kurongeka kwekoil conformation (Fig. 4b).Kemikari shanduko yekuongorora yakasaririra methionine iripo muNT chete yakaratidza kuti iyi domain yakashandurwa kuita β-sheet chimiro.Nguva-inotenderera ye NT mumhinduro yakaratidza kuderera kweyunifomu musimba rechiratidzo (Fig. 4c), uye yakasimba-state NMR yeNT hydrogels yakaratidza kuti zvizhinji zveNT zvakasara zvakashandurwa kuva β-sheet structures (Fig. 4d).Iko kuenderana kwe2Rep hakugone kutsanangurwa zvakasiyana nekuda kwemaitiro ekuunganidza.Zvisinei, iyo yakasimba mamiriro NMR spectra yeNTCT uye His-NT2RepCT hydrogels yakatarisa yakafanana zvikuru (Fig. 4b; Supplementary Fig. 6b), zvichiratidza kuti 2Rep yakapa zvishoma kune chikamu chechimiro cheHi-NT2RepCT hydrogel.Kune CT hydrogels, α-helices, β-mashizha, uye random helical secondary structures zvakawanikwa zviripo (Supplementary Fig. 6d).Izvi zvinoratidza kuti mamwe mativi eCT anoramba ari α-helices nepo mamwe achive β-mashizha.Saka, mhedzisiro yeNMR spectroscopy inoratidza kuti NT yakakosha pakugadzirwa kwehydrogel uye zvakare inoshandura kuita β-sheet conformation pakusanganiswa ne2Rep uye CT.Zvichienderana neizvi, isu munguva pfupi yapfuura takaona kuti amyloid spatial zippers inogona kuumba mune ese mashanu helices eiyo NT domain, uye iyo Waltz algorithm yakafanotaura nzvimbo yeamyloidogenic muhelix 1 (Fig. 4e).
2D spectra ye15N-HSQC 10 mg/mL His-NT2RepCT solution isati yasvika (blue) uye maawa gumi nemapfumbamwe mushure mekuita incubation (tsvuku) pa37°C.Michinjikwa yemunhu ega mutsvuku uye F24, G136, polyA mublue spectrum inotaridzwa nebhii rimwechete amino acid zviratidzo uye nhamba dzasara.Iwo maseti anoratidza kutsamira kwesimba rechiratidzo panguva yezvisaririra zvakasarudzwa kubva kuNT, 2Rep, uye CT domains.b Solid-state radiofrequency (RFDR) spectra ye His-NT2RepCT hydrogels.Kuwirirana kweCα / Cβ zvakasara zvakaonekwa muRFDR spectra zvakagadziriswa nekuenzanisa nemuenzaniso wepeptide kemikari kuchinja uye maitiro anobva kune nhamba82,83 uye zvimiro zvavo zvechipiri.SSB - bhandi rinotenderera.c One-dimensional spectra ye15N-HSQC 10 mg/mL NT mhinduro panguva yekuvharirwa pa37 °C kwemaawa makumi matatu nematanhatu.Iyo inset inoratidza volumetric intensity maringe nenguva.d Solid state RFDR spectra yeNT hydrogels.Iko kuwirirana kweCα / Cβ zvakasara uye zvimiro zvavo zvechipiri zvakaonekwa muRFDR spectra zvinoratidzwa.e Kubva paNT45.79 fibrillation propensity propensity kubva kuZipper database (https://services.mbi.ucla.edu/zipperdb/).Simba reRosetta rehwindo rekuchinja mheni yenzvimbo yehexapeptide inoratidzwa mu kcal/mol.Mabhawa matsvuku anoreva hexapeptides ane yakakwira fibrosis propensity (Rosetta simba pazasi -23 kcal/mol; pazasi pemutsara une doti).Mazariro egirinhi anoratidza zvidimbu zvine simba reRosetta pamusoro pechikumbaridzo uye nokudaro kashoma kuumba steric zipi.Zvimedu zvine proline zvakabviswa kubva pakuongorora (pasina makoramu).Makwere anoratidza nzvimbo dzeamyloidosis dzakafanotaurwa neWaltz algorithm81 (https://waltz.switchlab.org).Kutevedzana kwemaamino acid asara e NT iri pamusoro, uye mhando dzezvisaririra zvinowanikwa mu β yechipiri chimiro (yakatemerwa neiyo solid-state NMR spectroscopy) inoratidzwa mutsvuku.Nzvimbo dzeashanu dzeNT α-helices dzakadomwa se (H1-H5)28.
Pa pH <6.5, HT inodzikira, ichishingirira kupisa- kana urea-induced denaturation18.Kujekesa kuti NT dimerization uye kugadzikana zvinokanganisa sei gelation, mhinduro dzine 100 mg/ml NT dzakadzorwa papH 8, 7, uye 6 vachishandisa vial inversion test.NT samples incubated pa pH 8 uye 7 gelled mushure memaminitsi makumi matatu pa 37 ° C, asi pH 8 gel yakaramba yakajeka, nepo pH 7 gel yakaratidza mvura inooneka (Fig. 5a).Kusiyana neizvi, mhinduro ine HT papH 6 haina kuumba gel, uye mvura zhinji yaigona kuonekwa mushure memaminitsi makumi maviri pa37 ° C.Izvi zvinoratidza kuti dimers ivo pachavo uye / kana kugadzikana kwavo kwepamusoro zvichienzaniswa nemamonomers kudzivirira gelation.Kuumbwa kwekunaya kwe NT pa pH 7 ne 6 kwakanga kusingatarisirwi, sezvo zvichinzi NT inonyungudika pa 200 mg/ml27, inopeta nyore nyore mushure mekupisa kwekupisa, uye inochengeta α-helix pamutengo wakaderera we. pH 18. Tsananguro inogoneka yeiyi misiyano ndeyokuti ongororo dzakambotaurwa dzakaitwa pakamuri tembiricha kana pazasi, kana pane yakaderera mapuroteni concentrations16,18,19.
NT vial inversion test (100 mg/mL) papH 8, 7, 6 uye 154 mM NaCl (pH 8) mushure mekuita incubation pa37°C.b NT CD spectra ine uye isina 154 mM NaF uye 154 mM NaCl, zvichiteerana.Molar ellipticity pa222 nm inoshandurwa kuita chikamu chezvakasikwa zvakapetwa.c NT inversion assay (100 mg/mL) NT* (37 °C uye 60 °C), NTA72R (37 °C), uye His-NT-L6 (37 °C uye 60 °C).d CD chimiro che NT mutants NT*, NTA72R, uye His-NT-L6.Molar ellipticity pa222 nm inoshandurwa kuita chikamu chezvakasikwa zvakapetwa.e Inversion bvunzo yeNTFlSp, NTMiSp uye yakaderedzwa NTMiSp (100 mg/mL).Scale bar 5 mm.f CD spectra yeNT, NTFlSp, NTMiSp uye yakaderedzwa NTMiSp.Molar ellipticity pa222 nm inoshandurwa kuita chikamu chezvakasikwa zvakapetwa.Yakazara NT spectra pa25 °C uye 95 °C inoratidzwa muSupplementary Figure 8.
Physiological salt concentration inotaridza kudyidzana kwe electrostatic pakati pe NT subunits uye dimerization yeT kuendesa kune yakaderera pH18.Takaona kuti kuvapo kwe 154 mM NaCl uye NaF zvechokwadi inhibit gelation, maererano (Fig. 5a, b; Supplementary Fig. 2b) uye kuti iyi munyu yakawedzera kugadzikana kwekupisa kweNT monomers (Fig. 5b, Supplementary Fig. 8) .Iyo inoratidzawo kuti kugadzikana kwekusimudzira, pane dimerization, kunodzivirira kuumbwa kwegel.
Kuenderera mberi nekuongorora basa reprotein dimerization uye kugadzikana mugelation, takashandisa maviri mutants, NT * uye NTA72R, ayo zvakare anoramba ari monomeric pane yakaderera pH28.30.NT* ndeye kaviri kubhadharisa reversal mutant umo iyo inooneka dipolar charge kugovera kwemonomeri inogadzika, iyo inodzivirira dimerization uye zvakanyanya kuwedzera monomer kugadzikana.NTA72R inobhadhariswa dipole, asi Arg-inotsiva Ala iri pamuganho we dimer, saka shanduko dzinokanganisa kupindirana kwe subunit kunodiwa kuti dimerization.Pakunyura pa 37 ° C, NT * haina kuumba hydrogel, nepo NTA72R yakagadzira gel opaque kwemaminitsi gumi nemashanu (Fig. 5c).Sezvo zvose NT * uye NTA72R zvisingakwanisi kuderedza asi zvakasiyana mukugadzikana kwemonomeri (Fig. 5d), izvi zvinoguma zvinoratidza zvakasimba kuti kugadzikana kwepamusoro kwe thermodynamic kunodzivirira NT kubva kugelling.Izvi zvinotsigirwawo nechokwadi chokuti HT * inoumba gel kana isina kugadzikana pakupisa kwepamusoro (mushure me8 min pa60 ° C; Fig. 5c).Izvo zvakamboratidzwa kuti iyo yakakwira yemethionine muNT inonyungudutsa kupetwa kwayo kwechisikigo uye kuti matanhatu Met to Leu anotsiva (inonzi pano seYake-NT-L6) inodzikamisa zvakanyanya iyo NT46 monomer.Zvichienderana nefungidziro yekuti kuchinjika kwechimiro kunodiwa pakugadzira gel NT, takaona kuti His-NT-L6 yakagadzika mutant haina gel pa37 ° C (Mufananidzo 5c, d).Zvisinei, Yake-NT-L6 yakagadzirawo gel pamusoro pekugadzirisa pa 60 ° С kwemaminetsi makumi matanhatu (Fig. 5c).
Kugona kweNT kusandura kuita β-sheet zvimiro uye kuita mahydrogel anoita seanoshanda kune mamwe asi kwete ese eNT madomasi espidroin.NTs kubva kumhando dzakasiyana dzesirika nemhando dzedandemutande, Trichonephila clavipes (NTFlSp), yakagadzira gels zvisinei nekuti ine methionine yakadzikira uye kugadzikana kwakanyanya kwekushisa (Fig. 5e, f uye Supplementary Table 2).Kusiyana neizvi, NT kubva kudiki ampullar protein spidroin kubva kuAraneus ventricosus (NTMiSp) ine yakaderera thermal kugadzikana uye yakakwirira methionine zvinyorwa hazvina kuumba hydrogels (Supplementary Table 2 uye Fig. 5e, f).Iyo yekupedzisira inogona kubatanidzwa nekuvapo kwe intramolecular disulfide bonds29,47.Zvichienderana, apo zvisungo zve disulfide zve NTMiSp zvakaderedzwa, zvakagadzira hydrogel mushure mekuvhara pa 37 ° C kwemaminitsi gumi (Fig. 5e).Mukupedzisa, zvinofanirwa kucherechedzwa kuti kuchinjika kwechimiro kwakakosha, asi kwete chete, mucherechedzo wekugadzira gel kubva kuNT.Chimwe chinhu chingave chakakosha ndechekuita kuumba amyloid fibrils, uye kuongorora ne zipper dhatabhesi uye Waltz algorithm yakaratidza kuwirirana pakati pekugona kuumba gels uye kuvapo kweamyloidogenic matunhu, pamwe nehupamhi hwematunhu akafanotaurwa. kugadzira steric zippers.Pakanga pane kuwirirana (Supplementary Table 2 uye Supplementary Fig. 9).
Kugona kwetestamende itsva kugadzira fibrils uye kuumba gerosi pasi pemamiriro akanaka kwakaita kuti tifungidzire kuti kusanganiswa kweNT nemamwe mapuroteni zvimedu kunogona kuramba kuumba majiro ane basa rakazara remubatanidzwa wekusanganiswa.Kuti tiedze izvi, takaunza green fluorescent protein (GFP) uye purine nucleoside phosphorylase (PNP) paC-terminus yeNT, zvichiteerana.Mapuroteni emusanganiswa akaguma aratidzwa muE. coli ane goho rekupedzisira rakanyanya (150 mg/L uye 256 mg/L shake flask tsika dzeHis-NT-GFP neHis-NT-PNP, zvichiteerana), zvinoenderana nezvakaratidzwa. kune Mamwe mapuroteni akasanganiswa kuNT Ref.30. Yake-NT-GFP (300mg / mL) uye Yake-NT-PNP (100mg / mL) mapuroteni ekubatanidza akagadzira gels mushure meawa 2 uye maawa 6.5 pa37 ° C uye, zvakakosha, chikamu cheGFP chakaramba chisina kuchinja.yakacherechedzwa mushure mekugezera, ne> 70% yekutanga fluorescence intensity yakasara mushure megelation (Fig. 6a).Kuyera chiitiko chePNP mune yake-NT-PNP mhinduro uye gels, taifanira kudzikisa iyo fusion protein neNT nekuti iyo enzymatic chiitiko cheiyo yakachena gadziriro yaive kunze kweiyo yekuona huwandu hweyeyedzo pane gelling concentration.Iyo gel yakaumbwa nemusanganiswa ine 0.01 mg / mL Yake-NT-PNP uye 100 mg / mL NT yakachengetedza 65% yebasa rekutanga enzymatic ye preincubated samples (Fig. 6b).Iyo gel yakaramba yakasimba panguva yekuyera (Supplementary Fig. 10).
a Relative fluorescence intensity pamberi uye shure kwekugezwa kweHis-NT-GFP (300 mg/mL) uye inverted vial ine His-NT-GFP hydrogel (300 mg/mL) pasi pekuonekwa uye UV chiedza.Mapoinzi anoratidza kuyerwa kwemunhu (n = 3), mbariro dzekukanganisa dzinoratidza kutsauka kwakajairwa.Huwandu hwepakati hunoratidzwa pakati pezvikanganiso.b PNP basa rakawanikwa nekuongororwa kwefluorometric uchishandisa zvinogadzirisa uye gels zvinosanganisira NT (100 mg / ml) uye musanganiswa une 0.01 mg / ml yake-NT-PNP uye 100 mg / ml New Taiwan madhora.Iyo inset inoratidza vial yakapinduka ine hydrogel ine His-NT-PNP (5 mm scale bar).
Pano, tinoshuma kuumbwa kwema hydrogels kubva kuNT uye mamwe mapuroteni anonzi spidroin nekuisa mushonga weprotein pa 37 ° C (Mufananidzo 1).Isu tinoratidza kuti gelation inobatanidza nekushandurwa kwe-α-helices mu-β-layers uye kuumbwa kweamyloid-like fibrils (Fig. 3 uye 4).Kuwana uku kunoshamisa sezvo maNTs akamonerwa globular mashanu-helix masumbu anozivikanwa nekunyungudika kwakanyanya uye kugadzikana kwepamusoro pamisumbu>200 mg/mL pa4°C kwemazuva akati kuti27.Pamusoro pezvo, NTs inodzokorodza zviri nyore mushure mekupisa denaturation pane yakaderera mapuroteni emukati muµM.Maererano nemigumisiro yedu, kuumbwa kwefibril kunoda kusanganiswa kwe> 10 mg / mL protein concentration uye zvishoma zvishoma kutonhora (Fig. 1).Izvi zvinopindirana nepfungwa yokuti amyloid fibrils inogona kuumba kubva kune mapuroteni akapeta pasi rose ari mune imwe nzvimbo yakasununguka nekuda kwekuchinja kwekushisa pasi pemamiriro ehupenyu 48.Mienzaniso yemapuroteni anoshandura ichi anosanganisira insulin49,50, β2-microglobulin, transthyretin uye lysozyme51,52,53.Kunyange zvazvo NT iri α-helix munharaunda yayo, inenge 65% ye polypeptide chain inopindirana ne steric zipper formation (Fig. 4e) 45 .Sezvo monoma iri dynamically mobile46, inogona kufumura nzvimbo idzi dzinogona kuitika amyloidogenic pane tembiricha yakakwira zvine mwero uye pakukwira kwakanyanya kweprotein yakazara inogona kusvika padanho rakakosha reamyloid fibril formation54.Tichitevera kufunga uku, takawana kuwirirana kwakashata pakati pe spidroin concentration uye gelation nguva (Fig. 1c), uye kana monomeric NT conformation yakagadzikana kana nekuchinja (NT *, His-NT-L6) kana nekuwedzera munyu, inogona kudzivirira kuumba hydrogels (Fig. 5).
Muzviitiko zvakawanda, amyloid fibrils inonyangarika kubva pane mhinduro semvura, asi pasi pemamwe mamiriro ezvinhu vanogona kugadzira hydrogels55,56,57.Hydrogel-forming fibrils inowanzova nehupamhi hwepamusoro uye inoumba yakagadzikana matatu-dimensional network kuburikidza ne molecular entanglement,55,58 inoenderana nemhedzisiro yedu.Nokuda kwekugadzirwa kwe hydrogel in vitro, mapuroteni anowanzozarurwa zvakakwana kana kuti zvishoma, semuenzaniso, nekutsvaga kune organic solvents, kupisa kwepamusoro (70-90 ° C) uye / kana pasi pH (1.5-3.0) 59,60,61,62.Iyo spidroin hydrogels inotsanangurwa pano haidi kuomeswa nekugadziriswa, uye haidi kuyambuka-inobatanidza vamiririri kudzikamisa mahydrogel.
Izvo zvakambotaurwa kuti spidroin inodzokorora uye QDs, inoratidzika kunge ichipinda β-sheet switching panguva yekutenderera siriki, inoumba hydrogels.Tichienzanisa nezvatakawana, nguva dzeincubation uye/kana tembiricha dzekuisa dzaive dzakarebesa kana kukwira zvakanyanya, zvichiteerana, uye mahydrogel aikonzeresa aigara akajeka (Figure 7 uye Supplementary Table 1) 37, 38, 63, 64, 65, 66, 67, 68 , 69. Pamusoro pekutsanya gel nguva, NT hydrogels> 300 mg/mL (30%) yakapfuura mamwe ese anotsanangurwa anodzokorodza spider silk protein hydrogels, pamwe nemasikirwo emahydrogel akadai segelatin, alginate (2%), agar (0.5 % ) uye collagen.(0.6%) (Mufananidzo 7 uye Supplementary Tables 1 uye 3) 37,39,66,67,68,69,70,71,72,73,74.
Iyo nguva yegel uye elastic modulus ye hydrogels muchidzidzo ichi yakaenzaniswa nemamwe ma spidroin-based hydrogels uye akasarudzwa echisikigo hydrogel.Mareferensi anopiwa pamwe chete nekutsanangurwa kwemamiriro e gelation.APS Ammonium sulfate, tembiricha yekamuri.Data 37, 38, 39, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
Mhungu inoratidzika kunge yakagadzira nzira dzekudzivirira spidroin kubva mukugera panguva yekuchengetedza.Pasinei nehuwandu hwehuwandu hweprotein mumucheka wesilika, nzvimbo huru yekudzokorora yakabatana nedare rekupedzisira inoreva kuti kuonekwa kunooneka kweNT uye CT mugland inoenderana neanenge 10-20 mg / ml, pamuganhu wechidzidzo ichi.inodiwa kune in vitro yakacherechedzwa hydrogel kuumbwa.Mukuwedzera, kuenzana kwakafanana kwemunyu 16 kwakagadzikana NT, sezvinoitwa mumakirini esirika (Fig. 5b).NT conformation yakaongororwa muE. coli cytosol ikawanikwa yakapetwa zvakasimba kudarika kana ichiongororwa in vitro, zvichiratidza kuti munyu kana zvimwe zvinhu zvinodzivirira kuunganidzwa kwayo mu vivo.Zvakadaro, kugona kweNTs kusandura kuita β-sheet fibrils kungave kwakakosha pakuumbwa kwefilament uye kunofanirwa kuongororwa muzvidzidzo zvinotevera.
Mukuwedzera kune zvinyorwa zvitsva zve NT-amyloid-like fibril uye hydrogel formation yakaonekwa muchidzidzo ichi, tinoratidzawo kuti chiitiko ichi chinogona kunge chine biotechnological uye biomedical applications (Fig. 8).Sehumbowo hwepfungwa, takabatanidza NT neGFP kana PNP uye takaratidza kuti puroteni yekubatanidza inoumbawo mahydrogel kana yakavharwa pa37 ° C uye kuti zvikamu zveGFP nePNP zvinonyanya kuchengetedza basa ravo mushure megelation (Mufananidzo 6).Nucleoside phosphorylases akakosha catalysts synthesis ye nucleoside analogues75, izvo zvinoita kuti kuwanikwa kwedu kuve kwakakodzera kune iyo biopharmaceutical indasitiri.Pfungwa yekuratidzira mapuroteni ekubatanidza anoumba ma hydrogel akajeka pasi pemamiriro akanaka anobvumira kugadzirwa kweanoshanda ma hydrogel ane zvakanaka zvivakwa kune akasiyana siyana ekushandisa senge enzyme immobilization, inodzorwa kuburitswa kwezvinodhaka uye tishu engineering.Pamusoro pazvo, NT neNT* zvine hunyanzvi hwekutaura mamaki30, zvinoreva kuti NT nemhando dzayo dzinogona kushandiswa kugadzirwa kwepamusoro-soro kwemapuroteni emusanganiswa anonyungudika uye kugadzirwa kunotevera kwemapuroteni akananga mu3D hydrogel.
NT inonyungudika, α-helical uye yakagadzikana pakadzika pasi (µM) uye 37°C.Panguva imwecheteyo tembiricha, asi pakuwedzera kuwanda (> 10 mg/ml), NT inoumba gerosi dzinosanganisira amyloid-like fibrils.NT fusion mapuroteni anoumbawo fibrillar gels ane zvizere kushanda fusion zvimedu, achibvumira akasiyana mapuroteni kuti immobilized mu 3D hydrogels vachishandisa NT.Pasi: NT (PDB: 4FBS) nemifananidzo ye fiber network uye mapuroteni akabatanidzwa (anofungidzirwa uye asina kukweverwa kuchiyero, GFP PDB: 2B3Q, 10.2210/pdb2B3Q/pdb; PNP PDB: 4RJ2, 10.2210/pdb4RJ2).
Iwo anovaka (ona Supplementary Table 4 yerunyoro rwakakwana kusanganisira amino acid sequences) akaumbwa kuita plasmid pT7 uye akashandurwa kuita E. coli BL21 (DE3).E. coli ine engineered plasmids yakaiswa muLuria broth yakawedzerwa kanamycin (70 mg / l) uye yakakura usiku hwose pa30 ° C uye 250 rpm.Tsika yacho yakabva yaiswa 1/100 muLB medium ine kanamycin uye yakasimudzwa pa30 ° C uye 110 rpm kusvikira OD600 yasvika 0.8.Pazvidzidzo zveNMR, mabhakitiriya akakura muM9 mashoma epakati ane 2 g yeD-glucose 13C (Aldrich) uye 1 g yeammonium chloride 15N (Cambridge Isotope Laboratories, Inc.) yeprotein labeling ine isotopes.Dzikisa tembiricha kusvika pa20 madhigirii celsius uye kurudzira kutaura kweprotein ne 0.15 mM isopropylthiogalactopyranoside (final concentration).Mushure mehusiku mapuroteni kutaura, masero akakohwewa pa7278 × g, 4 ° C ye20 min.Masero pellets akamiswa zvakare mu20 mM Tris-HCl, pH 8, uye akaomeswa nechando kusvika kumwe kushandiswa.Masero akanyungudutswa akaiswa lysed achishandisa sero diruptor (TS akateedzera michina, Constant Systems Limited, England) pa30 kPa.Ipapo malysates akaiswa centrifuged pa25,000 g kwemaminitsi makumi matatu pa4 ° C.Kune NTMiSp, pellet yakabva yamiswa zvakare mu 2 M urea, 20 mM Tris-HCl, pH 8, uye sonicated for 2 min (2 s on/off, 65%), ndokuzoiswa centrifuged zvakare pa25,000 xg, 4° C. mukati 30 min.Iyo supernatant yakatakurwa panhoramu yeNi-NTA, yakagezwa ne20 mM Tris-HCl, 2 mM imidazole, pH 8, uye pakupedzisira protein yakaburitswa ne20 mM Tris-HCl, 200 mM imidazole, pH 8. Kugadzira NT2RepCT uye NTCT, thrombin digestion inosuma saiti (ThrCleav) pakati peYake neNT.Thrombin cleavage nzvimbo dziripowo muHis-NT-ThrCleav-2Rep (inogadzira 2Rep), His-thioredoxin-ThrCleav-NT (inogadzira NT), His-thioredoxin-ThrCleav-CT (inogadzira CT), His-Thioredoxin-ThrCleav-NT .* (inobudisa NT *), His-Thioredoxin-ThrCleav-NTA72R (inogadzira NTA72R), His-Thioredoxin-ThrCleav-NTFlSp (inobudisa NTF1Sp), uye His-Sulphur Redoxin-ThrCleav-NTMiSp (inobudisa NTMiSp).Izvo zvakagadziriswa zvakasvibiswa ne thrombin (1: 1000) uye dialyzed usiku hwose pa4 ° C. ne 20 mM Tris-HCl, pH 8, uchishandisa Spectra / Por dialysis membrane ine molecular weight threshold ye6-8 kDa.Mushure me dialysis, mhinduro inotakurwa pane Ni-NTA column uye tsvina ine protein yekufarira inounganidzwa.Kuwanda kweprotein kwakatemwa nekuyera kunyura kweUV pa280 nm uchishandisa kutsakatika kweprotein yega yega, kunze kweNTF1Sp, iyo yakashandisa iyo Bradford assay zvinoenderana neprotocol yemugadziri.Kuchena kwakatemwa neSDS polyacrylamide (4-20%) gel electrophoresis uye Coomassie brilliant blue staining.Mapuroteni akaiswa mukati mekushandisa centrifuge mafirita (VivaSpin 20, GE Healthcare) pa4000 xg ine 10 kDa molecular weight cutoff mumaminetsi makumi maviri.
Cheka mushonga weprotein uye unyatso pombi 150 µl mu 1 ml yakajeka septum vial (8 x 40 mm Thermo Scientific).Machubhu akavharwa uye akavharwa neparafilm kudzivirira kubuda.Mienzaniso (n = 3) yakavharwa pa 37 ° C kana 60 ° C uye nguva nenguva inverted kuti ione gelation.Samples dzisina gel dzakaiswa kweinenge svondo rimwe chete.Deredza NTMiSp disulfide bond ne 10 mM DTT pa 10 µM protein.Kuti uongorore kugezwa kwemachira edandemutande echisikigo, spider yebhiriji yeSweden yakachekwa, iwo maviri makuru maglands akaiswa mu200 μl ye20 mM Tris-HCl buffer pH 8 uye akachekwa kuti abvumire kupfekedza kuparadzaniswa kubva kune glands..Zviri mukati mematehwe zvinonyungudutswa mubuffer, 50 µl yekuona huremu hwakaoma (nekupfekedza mabhodhoro akazaruka pa60 °C kusvika kuhuremu hwakasimba) uye 150 µl yegelation pa37 °C.
Iyo yekuyeresa geometry/mudziyo inogadzirwa nesimbi isina tsvina uchishandisa parallel ndiro ine dhayamita yepamusoro ye20 mm uye gap re0.5 mm.Pisa muenzaniso kubva pa25 °C kusvika 45 °C uye kudzokera ku25 °C pamwero we1 °C paminiti uchishandisa simbi isina tsvina pasi Peltier plate.Vibrational zviyero zvakaitwa pafrequency ye 0.1 Hz uye mumutsara we viscoelastic nharaunda yezvinyorwa pane strain ye5% uye 0.5% yemasampuli e100 mg/mL uye 300-500 mg/mL, zvichiteerana.Shandisai humidity chamber kudzivirira kubuda.Data yakaongororwa pachishandiswa Prism 9.
Yekuunganidza infrared (IR) spectra pane tembiricha yekamuri kubva pa800 kusvika 3900 cm-1.Chishandiso cheATR, pamwe chete negwara rechiedza kuburikidza ne spectrometer, rinocheneswa nemhepo yakaoma yakasefa pamberi uye panguva yekuyedza.Mhinduro (500 mg / mL kuderedza kunyura kwemvura mu spectra) yakaputirwa pamakristasi, uye majiroji (500 mg / mL) akaumbwa asati ayera uye akazoendeswa kune makristasi (n = 3).1000 scans yakanyorwa nechisarudzo che 2 cm-1 uye zero duty cycle ye 2. Mutsara wechipiri wakaverengwa uchishandisa OPUS (Bruker) uchishandisa kutsetseka kwemazana mapfumbamwe.Iyo spectra yakajairwa kune imwechete yekubatanidza dunhu pakati pe1720 ne1580 cm-1 vachishandisa F. Menges "Spectragryph - Optical Spectroscopy Software".MuATR-IR spectroscopy, kudzika kwekupinda kwedanda re infrared mumuenzaniso kunotsamira nhamba yewave, zvichikonzera kunyura kwakasimba pazasi mawavenumber pane akakwira mawavenumber.Iyi mhedzisiro haina kugadziriswa kune iyo spectra inoratidzwa muFig.3 nekuti idiki kwazvo (Supplementary Fig. 4).Yakagadziriswa spectra yeiyi nhamba yakaverengerwa uchishandisa iyo Bruker OPUS software.
Muchidimbu, kuwanda kwakazara kweprotein conformations kunogoneka mushure mekutendeseka deconvolution yezvikamu mukati meamide I peak.Zvisinei, zvimwe zvipingamupinyi zvinomuka mukuita.Noise muspectrum inogona kuoneka se (enhema) peaks panguva yedeconvolution.Pamusoro pezvo, iyo yepamusoro nekuda kwekukotama kwemvura inopindirana nenzvimbo yeamide I peak uye inogona kunge iine hukuru hwakafanana hwemasampuli ane huwandu hwemvura, senge aqueous gel inodzidzwa pano.Nokudaro, hatina kuedza kuparadza zvachose amide I peak, uye zvatinoona zvinofanira kungotariswa mukutsigira dzimwe nzira dzakadai seNMR spectroscopy.
Mhinduro dze 50 mg/ml NT uye His-NT2RepCT dzakagadzirwa usiku hwose pa37°C.Iyo hydrogel yakabva yanyungudutswa ne 20 mM Tris-HCl (pH 8) kusvika kuhuwandu hwe 12.5 mg / ml, yakazununguswa zvakanaka uye yakaputirwa kuti iparadze gel.Zvadaro, iyo hydrogel yakaderedzwa ka10 ne20 mM Tris-HCl (pH 8), 5 μl yemuenzaniso yakashandiswa kune grid yemhangura yakavharwa neformvar, uye sampu yakawandisa yakabviswa nebepa rekudzima.Samples dzakagezwa kaviri ne5 µl yeMilliQ mvura uye yakasvibiswa ne1% uranyl formate kwemaminitsi mashanu.Bvisa tsvina yakawandisa nebepa rinopinza, wobva waomesa mhepo mesh.Kufungidzira kwakaitwa pamagridi aya uchishandisa FEI Tecnai 12 Spirit BioTWIN inoshanda pa100 kV.Mifananidzo yacho yakanyorwa pa x 26,500 uye x 43,000 magnifications uchishandisa Veleta 2k × 2k CCD kamera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).Pamuenzaniso wega wega (n = 1), 10-15 mifananidzo yakanyorwa.ImageJ (https://imagej.nih.gov/) yakashandiswa pakuongorora mifananidzo uye kuyerwa kwefiber diameters (n = 100, fibers dzakasiyana).Prism 9 yakashandiswa kuita unpaired t-test (two-tailed).Anoreva His-NT2RepCT neNT fibrils aive 11.43 (SD 2.035) uye 7.67 (SD 1.389) nm, zvichiteerana.Nguva yekuvimba (95%) ndeye -4.246 kusvika -3.275.madhigirii erusununguko = 198, p <0.0001.
80 µl yemasampuli emvura ane 10 µM thioflavin T (ThT) akayerwa nekatatu (n = 3) pasi pemamiriro akasimba achishandisa Corning 96-tsime dema pasi pezasi akajeka pasi mahwendefa (Corning Glass 3881, USA).Fluorescence misiyano yakanyorwa pachishandiswa 440 nm excitation sefa uye 480 nm emission sefa (FLUOStar Galaxy kubva kuBMG Labtech, Offenburg, Germany).Chiratidzo cheTT chakanga chisina kuzara kana kudzimwa, sezvo kuyedza kwakasiyana-siyana kweTT kwakaitwa pasina kushandura kusimba kwechiratidzo.Rekodha kunyura pa360 nm yekuyera kuyerwa kwemhute.Pakuedza kudyara mbeu, 100 mg/mL majirosi akaumbwa pa37° C., akamiswazve, uye akashandiswa kudyara pa molar ratios ye5%, 10%, ne20%.Data yakaongororwa pachishandiswa Prism 9.
Thaw stocks eHis-NT2RepCT uye NT>100 mg/mL paaizi uye kusefa kuburikidza ne0.22 µm sefa.Concentrations yakaverengerwa nekuyera kunyura pa280 nm uchishandisa Nanodrop.Mumatsime e96-tsime dema risingasviki ndiro (Corning) rine pasi rakajeka, sampuli dzakaderedzwa kusvika ku20 mg/ml mu20 mM Tris-HCl pH 8 uye yakasanganiswa ne5 μM ThT (final concentration), yakazara sampuli 50 μl vhoriyamu.Samples akafananidzirwa maminetsi ega ega e10 pa37 ° C pane CellObserver (Zeiss) microscope ine transmitted light channel uye FITC excitation uye emission filter seti yeThT imaging.A 20x/0.4 lens inoshandiswa pakufungidzira.Zen Blue (Zeiss) uye ImageJ (https://imagej.nih.gov/) zvakashandiswa pakuongorora mifananidzo.Gels dzakagadzirirwawo kubva kuNT uye His-NT2RepCT mhinduro pamusangano we 50 mg / mL ine 20 mM Tris pH 8 uye 5 µM ThT uye incubated pa 37 ° C ye 90 min.Zvidimbu zvegel zvakaendeswa kutsime idzva rine 20 mM Tris, pH 8, uye 5 μM ThT mundiro isina-inosunga dema 96 yakanyatsojeka yepasi ndiro.Wana girinhi fluorescence uye yakajeka munda mifananidzo pa20x/0.4 magnification.ImageJ yakashandiswa kuongorora mufananidzo.
Solution NMR spectra yakawanikwa pa310 K pa600 MHz Bruker Avance Neo spectrometer ine QCI Quadrupole Resonance Pulsed Gradient Field Cryoprobe (HFCN).NMR samples dzine 10 mg/mL homogeneous protein yakanyorwa ne13C, 15N, yakanyungudutswa mu20 mM Tris-HCl (pH 8), 0.02% (w/v) NaN3, 5% DO (v/v), (n = 1) .Kemikari kuchinja kweNT2RepCT papH 6.7 yakashandiswa kupa peak 23 mu 2D spectrum ye15N-HSQC.
Magic angle spinning solid NMR (MAS) spectra ye13C, 15N-yakanyorwa mahydrogels akanyorwa paBruker Avance III HD spectrometer pa800 MHz yakagadzirirwa 3.2 mm 13C/15N{1H} electronless probe.Sample tembiricha yaidzorwa pachishandiswa tembiricha tembiricha gasi rukova pa277 K. Two-dimensional dipole rotational resonance (DARR) 76 uye redhiyo frequency reconnection (RFDR) 77 spectra yakawanikwa paMAS frequencies ye12.5 kHz uye 20 kHz, zvichiteerana.Cross polarization (CP) kubva ku1H kusvika 13C yakaitwa uchishandisa mutsara rampu kubva pa60.0 kusvika 48.0 kHz pa1H, 61.3/71.6 kHz pa13C (pa12.5/20 kHz MAS) uye nguva yekubata 0.5–1 ms.Spinal6478 decoupling pa73.5 kHz yakashandiswa panguva yekuunganidza data.Nguva yekutora yaive 10 milliseconds uye kunonoka kutenderera kwaive 2.5 masekonzi.Iyo imwechete-yakabatana Cα / Cβ correlations yakaonekwa muRFDR spectra yakagoverwa zvichienderana nehunhu hwakasara-mhando yemakemikari shanduko uye kuwanda-kwakabatana kuwirirana muDARR spectra.
Iyo Zipper79 dhatabhesi (https://services.mbi.ucla.edu/zipperdb/) yakashandiswa kuongorora flutter tendencies uye Rosetta simba reNT, NTFlSp, uye NTMiSp.Iyo Zipper dhatabhesi inoverengera Rosetta Energy80, iyo inosanganisa akati wandei emahara simba mabasa kuenzanisira uye kuongorora mapuroteni chimiro.Chiyero chesimba che -23 kcal / mol kana pasi chinoratidza tsika yakakwirira ye fibrillate.Iyo yakaderera simba inoreva kugadzikana kwakawanda kwemaviri β-tambo mune zipper conformation.Pamusoro pezvo, iyo Waltz algorithm yakashandiswa kufanotaura amyloidogenic matunhu muNT, NTFlSp uye NTMiSp Ref.81. (https://waltz.switchlab.org/).
Mushonga weprotein weNT wakasanganiswa ne2-(N-morpholino)ethanesulfonic acid (MES) buffer papH 5.5 uye 6.0 kudzikisa pH kusvika pH 6 ne7, zvichiteerana.Mapuroteni ekupedzisira aive 100 mg / ml.
Kuyera kwakaitwa paJ-1500 CD spectrometer (JASCO, USA) vachishandisa 300 μL cuvette ine nzira yekuona ye 0.1 cm.Mapuroteni akaderedzwa kusvika ku10 μM (n = 1) mu20 mM phosphate buffer (pH 8).Kuti uongorore kugadzikana kweprotein muhupo hwemunyu, mapuroteni akaongororwa panguva imwechete (n = 1) mu 20 mM phosphate buffer (pH 8) ine 154 mM NaF kana NaCl, maererano.Kupisa kwekushisa kwakanyorwa pa 222 nm kubva ku25 ° C kusvika ku95 ° C nekupisa kwe1 ° C / min.Huwandu hwemapuroteni akapetwa akaverengerwa pachishandiswa fomula (KDmeasure - KDfinal)/(KDstart - KDfinal).Pamusoro pezvo, zviratidziro zvishanu zvakarekodhwa kune imwe neimwe sampuli kubva pa260 nm kusvika 190 nm pa25 ° C uye mushure mekupisa kusvika 95 ° C.Maonero mashanu akaenzana, akatsetseka uye akashandurwa kuita molar ellipticity.Data yakaongororwa pachishandiswa Prism 9.
The fluorescence intensity yeHis-NT-GFP (300 mg/mL, 80 µL) yakayerwa nekatatu (n = 3) mu96-tsime Corning mahwendefa ane dema rakajeka pasi (Corning Glass 3881, USA) pasi pemamiriro akasimba.Yera masampuli ane fluorescence-based plate reader ine excitation wavelength ye 395 nm uye rekodha kubuda kwacho pa509 nm isati yaiswa gelation uye maawa maviri gare gare pa37°C.Data yakaongororwa nePrism 9.
Purine nucleoside phosphorylase basa rekuongorora kit (fluorometric nzira, Sigma Aldrich) yakashandiswa maererano nemirairo yemugadziri.Kuyera chiitiko mumajeri uye zvigadziriso zvine His-NT-PNP, sanganisa 10 ng yeYake-NT-PNP ne100 mg/mL NT kusvika kuhuwandu hwe2 µL nekuti gel yakapa chiratidzo pamusoro penguva yekuona yeti.Zvidzoreso zvegel uye zvigadziriso pasina Yake-NT-PNP zvakasanganisirwa.Izvo zviyero zvakaitwa kaviri (n = 2).Mushure mekunge chiitiko chacho chayerwa, musanganiswa wekuita wakabviswa uye gel rakatorwa mifananidzo kuti ive nechokwadi chekuti gel yakaramba yakasimba panguva yekuyera.Data yakaongororwa pachishandiswa Prism 9.
Kuti uwane rumwe ruzivo nezve dhizaini yekudzidza, ona Nature kudzidza abstract yakabatana nechinyorwa ichi.
Mifananidzo 1 uye 2 inopa data yekutanga.1c, 2a–c, 3a, b, e–g, 4, 5b, d, f, uye 6, Supplementary Figs.3, muonde wekuwedzera.5a, d, muonde wekuwedzera.6 uye muonde wekuwedzera.8. Data Data kubva kuchidzidzo ichi inoiswa mudura reZenodo https://doi.org/10.5281/zenodo.6683653.Iyo NMR data yakawanikwa muchidzidzo ichi yakatumirwa kuBMRBig repository pasi peiyo ID bmrbig36 yekupinda.Zvimiro zveGFP nePNP zvakatorwa kubva kuPDB (GFP 2B3Q, PNP 4RJ2).
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