310 10*1mm Insimbi engagqwali ehlanganisiwe yengxenye yamakhemikhali yamashubhu, Izizinda ze-N-terminal ze-spidroin zakha ama-hydrogel asekelwe kuma-amyloid fibrils futhi zinikeza inkundla yokuvinjelwa kwamaprotheni.

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Ukucaciswa

310 10*1mm abahlinzeki beshubhu bensimbi engagqwali abahlanganisiwe

Ukwakheka Kwamakhemikhali

Izakhiwo Zemishini

Amaprotheni kasilika wesicabucabu (amaprotheni kasilika wesicabucabu) anemisebenzi eminingi engaba khona ekuthuthukisweni kwezinto ezintsha ze-biomaterials, kodwa imvelo yawo ejwayele ukuhlanganisa izinto eziningi kanye ne-aggregation iwenza kube nzima ukutholakala futhi kulula ukuwasebenzisa.Lapha sibika ukuthi amaprotheni e-spidroin amancane aphinde ahlanganiswe futhi, okubalulekile, isizinda se-N-terminal (NT) ngokwaso sakha ngokushesha ama-hydrogel azisekelayo futhi abonisa ngale ku-37 °C.amaprotheni e-fusion ahlanganisa i-NT kanye ne-green fluorescent protein noma i-purine nucleoside phosphorylase yakha amaprotheni e-fusion asebenza ngokugcwele.Ama-Hydrogel.Imiphumela yethu ikhombisa ukuthi amaprotheni ahlanganisiwe we-NT kanye namaphrotheni e-fusion anikeza isivuno esikhulu sokubonisa futhi anikeze ama-hydrogel ngezakhiwo ezikhangayo njengokusobala, i-gelation ngaphandle kokuxhumanisa, kanye nokunganyakazisi okuqondile kwamaprotheni asebenzayo ekumineni okuphezulu.
Izicabucabu zinezindlala zikasilika ezifinyelela kweziyisikhombisa, ngayinye ikhiqiza uhlobo oluthile lukasilika.Zonke izinhlobo zikasilika eziyisikhombisa zakhiwe ngamaprotheni kasilika wesicabucabu (ama-spidroins) cishe izinsalela eziyizi-6000 ubude futhi aqukethe indawo enkulu yokuphinda emaphakathi ezungezwe izizinda eziyindilinga ze-N- kanye ne-C-terminal (NT kanye ne-CT)1,2.Uhlobo lukasilika olufundwa kabanzi, i-primary ampulla, lukhiqizwa indlala eyinhloko ye-ampulla.Kule ndlala, i-monolayer yamaseli e-epithelial ihlanganisa amaprotheni e-spidroin futhi iwafihle kulumen yendlala, lapho ekhona ngendlela encibilikayo (i-doping) ekugxilweni okuphezulu kakhulu (30-50% w/v)3,4.Ukuhleleka nokuvumelana kwamaprotheni e-ampullar spidroin ayinhloko ku-gland kuye kwaphikiswana ngakho, kodwa ubufakazi obuningi bokuhlola bubonisa ukuba khona kwe-helical ngokuvamile kanye / noma okungahleliwe kwe-helical conformation kanye nezakhiwo ze-micellar noma i-lamellar5,6,7,8,9,10.Nakuba izizinda eziphindaphindayo zilawula izakhiwo zemishini yezintambo zesilika, zakha i-β-sheet nanocrystals kanye nezakhiwo ze-amorphous11,12,13,14,15, izizinda zokugcina zilawula izintambo ze-silika ekuphenduleni izimo ezishintshayo eduze kwe-silk gland16,17,18.Ngokulawula ukwakheka kukasilika, 19. Izizinda zetheminali zigcinwa ngokuziphendukela kwemvelo futhi umsebenzi wazo ungase ufane nawo wonke amaprotheni e-spidroin 2,20,21.Ngesikhathi sokudlula endlaleni, i-pH ye-spidroin iyehla isuka ku-7.6 iye ku-<5.716 futhi iyanda ngokushear nokunwebeka okuxhunyaniswa nokunyakaza ngomgudu oncipha kancane kancane.Esixazululweni, i-CT iyi-α-helical constitutive parallel dimer17, kodwa ngokuphendula amandla aphansi we-pH namandla e-shear, i-CT ivula futhi ishintshe u-β-layers16, 17, okungenzeka ukuthi iqalise izendlalelo ze-β ezindaweni eziphindaphindiwe ze-Convert 16. I-NT i-monomeric ngaphansi izimo ezibonisa izimo ku-lumen ye-gland futhi zilamula ukunyibilika kwe-spidroin, kodwa nge-pH encishisiwe, ukuvela kwenani lamaketanga aseceleni e-carboxylic acid kuholela ekuncipheni kwe-NT nge-pKa ecishe ibe ngu-6.5, ngaleyo ndlela kusimamise i-NT futhi kulungiswe i-spidroin enkulu. amanani.amanethiwekhi16,18.Ngakho-ke, i-NT idlala indima ebalulekile ekwakhiweni kwe-filament, ishintsha isuka ku-monomer ekugqokeni ibe yi-dimer ku-fiber23,24,25.I-NT isalokhu incibilika kakhulu futhi i-helical ngaphansi kwazo zonke izimo ezifundwe kuze kube manje16, 18, 19, 20, 26, 27, 28, 29, okugqugquzele ukuthuthukiswa kwayo njengelebula elithuthukisa ukunyibilika kokukhiqizwa kwamaphrotheni e-heterologous.
I-recombinant mini spider silk protein, ehlanganisa i-NT eyodwa, isifunda esisodwa esifushane, i-CT eyodwa, kanye ne-His-NT2RepCT tag (His-NT2RepCT) yokuhlanzwa, incibilika ku-aqueous buffer njengeprotein yesicabucabu sikasilika futhi ilingisa izici zomdabu ezibalulekile zesicabucabu sikasilika. .ukusakazwa 25.31.I-His-NT2RepCT ingaphothwa ibe imicu eqhubekayo kusetshenziswa umshini we-biomimetic lapho i-pH 8 encibilikayo ifakwa kubhavu wamanzi we-pH 525,32,33,34,35.Ukuvutshelwa kwe-Bioreactor ye-E. coli eveza i-His-NT2RepCT kanye nokwelashwa okwalandela ngemva kokwelashwa kubangele >14 g/L isivuno ngemva kokucwengwa.Isivuno esiphezulu, ukunyibilika okuphezulu, kanye nokuphendula okwanele kwe-His-NT2RepCT ezimeni ezine-acidic konke kubalulwe ku-NT23, 25, 34.
Lapha sibika ukwakheka okusheshayo kwama-hydrogel abonakalayo avela kumaprotheni e-spidroin aphinde ahlanganiswe, okuhlanganisa i-NT iyodwa, ngokufukamela isisombululo seprotheni ku-37 °C.Sisebenzisa i-thioflavin T fluorescence (ThT), i-Fourier iguqula i-infrared spectroscopy (FTIR), i-nuclear magnetic resonance spectroscopy (NMR) kanye ne-electron microscopy transmission (TEM), sithole ukuthi amaprotheni e-NT kanye ne-microspider ashintsha isakhiwo abe amashidi e-β namafayili afana ne-amyloid. lapho kwakhiwa ama-gel.Ngaphezu kwalokho, amaprotheni ahlanganisiwe we-NT kanye neprotheyini ye-fluorescent eluhlaza (GFP) noma i-purine nucleoside phosphorylase (PNP) enza ama-hydrogel anezingcezu ze-fusion ezisebenza ngokugcwele.Ukuvezwa komphumela ophezulu kubasingathi be-heterologous, kuhambisana nokwakheka okusheshayo kwama-hydrogel ngaphansi kwezimo zomzimba, kuvula ithuba lokukhiqizwa okungabizi kakhulu kwama-hydrogel anemisebenzi yobunjiniyela.
Ngokungafani namaphrotheni amaningi e-spidroin abikiwe kabusha, i-His-NT2RepCT izinzile kusibhafa se-Tris-HCl ku-pH 8 futhi ingagxiliswa kufika ku-500 mg/mL ngaphandle kwemvula25.Ngakho-ke, samangala ukuthola ukuthi le phrotheni yenza ngokushesha ama-hydrogel acacile, azisekela lapho efukanyelwa ku-37 ° C (Fig. 1b-d).Ucwaningo olwengeziwe lubonise ukuthi i-gelation ye-His-NT2RepCT yenzeke phezu kohlu olubanzi lwe-protein concentrations (10-300 mg / mL) nokuthi lokhu kuhlushwa kwakuhlotshaniswa ngokuphambene nesikhathi se-gelation (Fig. 1c kanye ne-Supplementary Fig. 1).Ukuthola ukuthi yiziphi izingxenye ze-His-NT2RepCT mediate hydrogel formation, sibe sesihlola isizinda ngasinye ngazinye nezinhlanganisela ezahlukene sisebenzisa i-flask inversion assay (Umfanekiso 1a,b).Zonke izingxenyana ezihloliwe ze-spidroin ehlangene zenze ama-gel (ekuhlanganiseni kweprotheyini engu-300 mg/mL) ngaphansi kwehora elingu-1, ngaphandle kwe-2Rep (Fig. 1b).Lokhu kuphakamisa ukuthi i-NT ne-CT iyodwa, ngokuhlangene, noma ehlotshaniswa nokuphindaphinda, ingakwazi i-gel ku-37 ° C nokuthi ithegi ye-His6 ayithinti le nqubo kunoma yiliphi izinga elibalulekile.Uma kubhekwa umbono ojwayelekile wokuthi i-NT iyiphrotheni encibilika kakhulu futhi ezinzile, nokuthi imibiko yangaphambilini ye-recombinant spidroin hydrogels iveze ukuthi imiphumela ye-gelation ihlobene nezinguquko eziguqukayo ezifundeni eziphindayo kanye/noma ama-CTs, i-NT ngokwayo ingenzeka.Ukutholakala kwe-gelation bekungalindelekile.Ithebula Lokwengeza 1) 37, 38, 39. Ngokuphawulekayo, i-NT isivele ifakwe phakathi kwemizuzu ye-10 ekuhlanganiseni kwe-≥ 300 mg / mL (Fig. 1c).Ukuhlolwa kwe-vial inversion ngokugxila okuhlukahlukene kwe-NT kubonise ukuthi ku->50 mg/mL isixazululo se-NT sigeze ngokushesha kune-His-NT2RepCT ekugxiliseni okuhambisanayo (w/v, Figure 1c).
Ukumelwa okuhleliwe kwezakhi ezihlukahlukene ze-spidroin ezifundwe kulo msebenzi.b Isikhathi sejeli singu-37 °C samaprotheni e-spidroin ahlukahlukene (300 mg/mL) aqinisekiswa ngokujikisa ibhodlela.Ijeli ye-CT ngokushesha ngaphandle kokufukamela (<300 mg/mL), i-2Rep precipitates (300 mg/mL, 5 mm isikali).c Isikhathi sejeli se-His-NT2RepCT ne-NT ekugxilweni kwamaprotheni okubonisiwe ku-37°C.d Izithombe zama-hydrogel e-His-NT2RepCT kanye ne-NT anespider kanye nohlamvu “NT” aphrintwe ngaphansi, ngokulandelana (kokubili 200 mg/mL, ibha yesikali 5 mm).
Ama-Hydrogel akhiwa amaprotheni e-spidroin ahlukahlukene anemibala ehlukene kancane, futhi ukubuka kwamehlo anqunu kubonisa amazinga ahlukahlukene okukhanya (Fig. 1b).Amajeli e-NT acace ngendlela emangalisayo kuyilapho amanye amajeli eba opaque.Amajeli e-His-NT2RepCT kanye ne-NT aphonswe kumashubhu angama-cylindrical angasuswa esikhunjeni esingaguquki (Fig. 1d).
Ukuze kuhlolwe ukuthi ingabe ijeli yemvelo yesicabucabu sikasilika ngaphansi kwezimo manje etholakala ibangela ukugeleza kwamaprotheni e-spidroin aphinde ahlanganiswe, kwaqoqwa izimbotshana ku-ampulla gland enkulu yesicabucabu sebhuloho laseSweden (Larinioides sclopetarius).Ama-coatings agcinwe ku-20 mM Tris-HCl kubhafa ku-50 mg/mL (ngokusekelwe esisindweni esomile esilinganisiwe), kodwa akukho ukugeleza okuye kwabonwa phakathi nezinsuku ezingama-21 zokufukamela ku-37 °C (Umfanekiso Owengeziwe 2a).
Ukuze ulinganise lawa ma-gel, izilinganiso ze-rheological zingasetshenziswa ukutadisha inqubo ye-gelation futhi inqume izici eziphelele zemishini.Ikakhulukazi, ukuqapha i-modulus yokugcina (i-elasticity) emazingeni okushisa aphakeme kunganikeza ulwazi mayelana nokushisa kwe-gelling kanye nezakhiwo ze-viscoelastic ze-coating.Ukuhlolwa kokukhuphuka kwezinga lokushisa (kusetshenziswa u-1°C/min ku-25-45°C, okusekelwe ocwaningweni lwangaphambilini kusetshenziswa izixazululo zesitoko sikasilika semvelo)40,41 kubonise ukuthi imoduli yokugcina yezixazululo ze-His-NT2RepCT ne-NT yenyuke ngokunyuka kwezinga lokushisa.yanda (Fig. 2 kanye Supplementary Fig. 3).Ngokuphawulekayo, imojula ye-NT yaqala ukukhula ngezinga lokushisa eliphansi uma liqhathaniswa ne-His-NT2RepCT, elihambisana nesikhathi sejeli esisheshayo esibonwa lapho i-NT ifakwe ngokuqondile ne-His-NT2RepCT ku-37 ° C (Umfanekiso 1).Ngemuva kokwehla kwezinga lokushisa okulandelayo, i-modulus yesitoreji ayizange ibuyele kumanani aphansi futhi yahlala ingaphezulu kwe-modulus yokulahlekelwa (bona i-Supplementary Fig. 3), ekhombisa i-gelation eqinile engenakuhlehliswa.Ngemuva kokufakwa kwe-gelation, i-elastic modulus yokugcina yayisukela ku-15 kuya ku-330 kPa kuma-hydrogel e-His-NT2RepCT ekuhlanganiseni kuka-100–500 mg/mL, kanye ne-elastic modulus yokugcina yama-hydrogel e-NT (100–500 mg/mL) asukela ku-2 kuye ku-1400. kPa (Umfanekiso, 2 kanye nedatha ephelele yerempu) bheka I-Supplementary Fig. 3).
Ukushintsha kwezinga lokushisa phakathi nezilinganiso ze-His-NT2RepCT (300 mg/mL) kanye ne-b NT (300 mg/mL) ngokuthuthumela.Imicibisholo ikhombisa ithrendi yezinga lokushisa, futhi ukufiphala okulula kwedatha yemojuli yesitoreji kubonisa ukuhlolwa ngamavelu etorque aphansi wensimbi kunaleyo eshiwo umenzi, okuyimbangela yomsindo owandayo.c Ukuqoqwa kwemojula yokugcina ye-His-NT2RepCT ne-NT ngemva kwezinga lokushisa eliphakanyisiwe (100, 300, kanye no-500 mg/mL).Konke ukufundwa kwamamojula kuthathwa ngemvamisa ye-0.1 Hz.
Njengendlela engaba khona yokuphenya izinguquko ezihambisanayo ezihlotshaniswa ne-gelation, siqophe i-spectra ye-FTIR ye-His-NT2RepCT ne-NT ngaphambi nangemuva kwe-gelation ku-37°C (Umfanekiso 3a,b).Njengoba bekulindelekile, i-spectra yezixazululo ze-His-NT2RepCT ne-NT zihambisana namaprotheni abonisa isakhiwo sesibili sekhoyili ye-α-helix/okungahleliwe, enebhendi ephinyiselwe ku-1645 cm-1.Kuwo womabili ama-hydrogel, i-gelation ibangele ukwakheka kwezingalo ezimbili phakathi kwebhande le-I cishe ngo-1617 cm-1 no-1695 cm-1 (Fig. 3a, b), okubonisa ukwakheka kwezakhiwo ze-antiparallel β-sheet.Lezi zinguquko zingabonakala ngokucacile ku-derivative yesibili efanele kanye nomehluko we-gelation spectra (I-Supplementary Fig. 4b).Amabhendi amabili e-NT β-layer agqame kakhulu kunalawo e-His-NT2RepCT, okubonisa ukuthi ingqikithi yokuqukethwe kwamabhendi we-β-layer ku-NT hydrogel yayiphezulu kunaleyo ye-NT2RepCT hydrogel.
i-spectra yokumunca ye-FTIR ye-His-NT2RepCT kanye ne-b NT (kokubili i-500 mg/mL) ngaphambi (isixazululo) nangemuva (ijeli) yokufakwa ku-37°C.c Izithombe ze-TEM zamajeli angu-50 mg/ml e-NT2RepCT amiswe kabusha kanye ne-d NT.Ibha yesikali engu-200 nm.e Fiber diameters of His-NT2RepCT and NT hydrogels.n = 100 ama-fibrils alinganisiwe, p <0.0001.Amabha wephutha abonisa ukuchezuka okujwayelekile.Isikhungo sebha yephutha sisho.Ukuhlolwa kuka-t okungabhanqiwe (okunemisila emibili) kusetshenziswe ekuhlaziyweni kwezibalo.f I-ThT fluorescence yamaprotheni e-spidroin ahlukahlukene (100 mg/mL) ku-37 °C ngaphandle kokuzamazama.g Ukuhlolwa kokujova kwe-NT (100 mg/mL) kusuka ku-100 mg/mL ijeli ye-NT eno-0%, 5%, 10%, kanye nembewu engu-20%.
Ukuhlaziywa kwejeli kusetshenziswa i-transmission electron microscopy (TEM) kubonise ukuthi i-hydrogel iqukethe ama-amyloid-like fibrils (Fig. 3c, 3d).Ama-fibril akhiwe nge-NT ayenwetshiwe (ama-5-12 nm ububanzi) futhi angenawo amagatsha, kuyilapho ama-fibril akhe-NT2RepCT ayemafushane ngobude futhi ebanzi kakhulu ngobubanzi (7-16 nm) (Fig. 3e).Le miphumela isivumele ukuthi silandele i-kinetics ye-fibrosis sisebenzisa i-thioflavin T (ThT) assay.Kuwo wonke ama-recombinant spidroin proteins, isignali ye-fluorescent yanda lapho amasampula efukanyelwa ku-37 °C (Fig. 3f, Supplementary Fig. 5a).Ngokuvumelana nalokhu kutholwe, ukuhlolwa kwe-microscopic ye-NT kanye ne-His-NT2RepCT ngaphansi kwezimo ze-gelling kwembule ukwanda okufanayo kwe-ThT fluorescence ngaphandle kokuqoqwa kwendawo okubonakalayo kwama-aggregates e-ThT-positive (I-Supplementary Fig. 5b, c).Ukwakhiwa kwe-fibrils ye-ThT-positive akuzange kuhambisane nokwanda kwe-NT kanye ne-His-NTCT turbidity (I-Supplementary Fig. 5d), okusho ukuthi inethiwekhi ye-fibrils ku-gel ingakha ngaphandle kokuyekethisa ukucaca kwe-gel.Ukuhlwanyela imbewu ngokungeza amanani amancane ama-fibril akhiwe ngaphambili kungasheshisa kakhulu ukwakheka kwe-fibril kwamanye ama-amyloids42,43,44 kodwa kwengeze u-5%, 10% noma 20% (w/w) NT esixazululo se-NT hydrocoagulants.imbewu umphumela (Fig. 3g).Mhlawumbe lokhu kungenxa yokuthi ama-fibrils ku-hydrogel alinganiselwe futhi awakwazi ukusetshenziswa njengembewu.
Ukuziphatha okungalindelekile kwamaphrotheni e-spidroin aphinde ahlangana emazingeni okushisa aphezulu kubangele olunye ucwaningo lwe-spectroscopy ye-nuclear magnetic resonance (NMR) ukuze kuhlonzwe izinguquko ezihambisanayo ezihambisana nokwakheka kwejeli.I-spectra ye-NMR yezixazululo ze-His-NT2RepCT ezirekhodwe ngokuhamba kwesikhathi ku-37°C ibonise ukuthi i-CT yayisagoqwe ngokwengxenye, kuyilapho amasiginali we-NT kanye ne-2Rep anyamalele (Fig. 4a), okuphakamisa ukuthi ngokuyinhloko kwakuyi-NT kanye ne-2Rep eyayilawula ukwakheka kwe-His- I-NT2RepCT i-hydrogel.Isignali ye-CT iphinde yehliswa ku-20% wokuqina kwayo kwasekuqaleni, okuphakamisa ukuthi i-CT nayo igxilile kakhulu futhi ifakwe esakhiweni se-hydrogel.Engxenyeni encane ye-CT, ehamba njengesampuleni efakwe ngaphambili futhi ngaleyo ndlela ibonwe yisixazululo se-NMR, isibuko sishoda amasiginali ezinsalela ezihleliwe eziyi-10 zokuqala, okungenzeka ngenxa yokunganyakazi okunzima kwengxenye enamathiselwe ye-His-NT2Rep .I-spectra ye-NMR ye--state of hydrogels -NT2RepCT yembula ukuba khona okuvelele kwe-α-helices kanye ne-β-layer futhi, ngezinga elincane, ukuguqulwa kwekhoyili okungahleliwe (Fig. 4b).Ukuhlaziywa kokushintshwa kwamakhemikhali kwezinsalela ze-methionine ezitholakala kuphela ku-NT kubonise ukuthi lesi sizinda siguqulelwe ekubeni isakhiwo seshidi le-β.I-spectra encike esikhathini se-NT esixazululweni ibonise ukwehla okufanayo ekuqineni kwesignali (Fig. 4c), kanye ne-solid state NMR ye-NT hydrogels ibonise ukuthi izinsalela eziningi ze-NT zaguqulwa zibe izakhiwo ze-β-sheet (Fig. 4d).Ukuhambisana kwe-2Rep akukwazanga ukunqunywa ngokuhlukile ngenxa yokuthambekela kwayo ekuhlanganiseni.Kodwa-ke, i-spectra ye-NMR yesimo esiqinile ye-NTCT kanye ne-His-NT2RepCT hydrogels ibukeka ifana kakhulu (Fig. 4b; I-Supplementary Fig. 6b), ephakamisa ukuthi i-2Rep inikele kancane engxenyeni yesakhiwo se-His-NT2RepCT hydrogel.Kuma-hydrogel e-CT, ama-α-helices, ama-β-sheets, kanye nezakhiwo zesibili ze-helical ezingahleliwe zitholakale zikhona (I-Supplementary Fig. 6d).Lokhu kuphakamisa ukuthi ezinye izingxenye ze-CT zihlala zingama-α-helices kuyilapho ezinye ziba amashidi angu-β.Ngakho, imiphumela ye-NMR spectroscopy iphakamisa ukuthi i-NT ibalulekile ekwakhekeni kwe-hydrogel futhi iphinde iguqule ibe ukuhambisana kwe-β-sheet ekuhlanganisweni ne-2Rep ne-CT.Ngokuvumelana nalokhu, sisanda kuthola ukuthi iziphuphu ze-amyloid zendawo cishe zakha kuwo wonke ama-helice amahlanu esizinda se-NT, futhi i-algorithm ye-Waltz yabikezela isifunda se-amyloidogenic ku-helix 1 (Fig. 4e).
I-spectra ye-2D ye-15N-HSQC 10 mg/mL Isixazululo se-His-NT2RepCT ngaphambi (okuluhlaza okwesibhakabhaka) namahora angu-19 ngemva kokufukamela (okubomvu) ku-37°C.Iziqongo eziphambanayo ngazinye ku-spectrum ebomvu kanye ne-F24, G136, polyA ku-spectrum eluhlaza okwesibhakabhaka kuboniswa ngohlamvu olulodwa lwezimpawu ze-amino acid nezinombolo ezisele.Amasethi abonisa ukuncika kokuqina kwesignali ngesikhathi ezinsalela ezikhethiwe kusukela kusizinda se-NT, 2Rep, ne-CT.b I-spectra ye-Solid-state radiofrequency (RFDR) yama-hydrogel e-His-NT2RepCT.Ukuxhumana kwezinsalela ze-Cα/Cβ ezibonwe ku-spectra ye-RFDR kwanqunywa ngokuqhathaniswa namashifu amakhemikhali e-peptide eyimodeli kanye namanani asuselwe kwizibalo82,83 kanye nezakhiwo zawo zesibili.I-SSB – ibhande eseceleni elijikelezayo.c I-spectra ye-One-dimensional ye-15N-HSQC 10 mg/mL NT isisombululo ngesikhathi sokufukamela ku-37 °C amahora angu-36.Isingeniso sibonisa ukushuba kwevolumu uma kuqhathaniswa nesikhathi.d Isimo esiqinile se-RFDR spectra sama-hydrogel e-NT.Ukuhlobana kwezinsalela ze-Cα/Cβ kanye nezakhiwo zazo zesibili ezibonwa ku-spectra ye-RFDR kubonisiwe.e Isekelwe kuphrofayela ye-NT45.79 ye-fibrillation propensity kusukela kusizindalwazi se-Zipper (https://services.mbi.ucla.edu/zipperdb/).Amandla e-Rosetta ewindi lokushintsha kombani we-hexapeptide aboniswa ku-kcal/mol.Amabha abomvu asho ama-hexapeptide ane-fibrosis propensity ephezulu (amandla e-Rosetta angaphansi -23 kcal/mol; ngaphansi komugqa onamachashazi).Amabha aluhlaza akhombisa izingcezwana ezinamandla e-Rosetta ngaphezu komkhawulo ngakho-ke mancane amathuba okuthi zenze iziphu eziqinile.Izingcezu eziqukethe i-proline azifakwanga ekuhlaziyweni (ngaphandle kwamakholomu).Izikwele zibonisa izindawo ze-amyloidosis ezibikezelwe i-algorithm81 ye-Waltz (https://waltz.switchlab.org).Ukulandelana kwezinsalela ze-amino acid ye-NT kuphezulu, futhi izinhlobo zezinsalela ezitholakala esakhiweni sesibili esingu-β (enqunywa i-spectroscopy ye-NMR yesimo esiqinile) ziboniswa ngokubomvu.Izikhundla ze-NT α-helices ezinhlanu zikhethwe ngokuthi (H1-H5)28.
Ku-pH <6.5, i-HT iyancipha, imelana nokushisa- noma i-denaturation eyenziwe nge-urea18.Ukuze kucaciswe ukuthi i-NT dimerization nokuzinza kuyithinta kanjani i-gelation, izixazululo eziqukethe i-100 mg/ml NT zazilawulwa ku-pH 8, 7, no-6 kusetshenziswa ukuhlolwa kwe-vial inversion.Amasampula e-NT afakwe ku-pH 8 kanye ne-7 afakwe ngemva kwemizuzu engama-30 ku-37 °C, kodwa ijeli ye-pH 8 yahlala icacile, kuyilapho ijeli ye-pH 7 ikhombisa imvula ebonakalayo (Fig. 5a).Ngokuphambene, isixazululo esiqukethe i-HT ku-pH 6 asizange senze ijeli, futhi imvula enkulu ingabonakala ngemva kwamaminithi angu-20 ku-37°C.Lokhu kuphakamisa ukuthi ama-dimers ngokwawo kanye/noma ukuzinza kwawo okuphezulu uma kuqhathaniswa nama-monomers avimbela i-gelation.Ukwakhiwa kwemvula ye-NT ku-pH 7 no-6 bekungalindelekile, njengoba kuye kwabikwa ukuthi i-NT ixubeka ku-200 mg/ml27, iphinda iphindeke kalula ngemva kokukhishwa kokushisa, futhi igcina i-α-helix ngamavelu aphansi i-pH 18. Incazelo engaba khona yalokhu kuhluka ukuthi ukuhlola okubikwe ngaphambilini kwenziwe ezingeni lokushisa legumbi noma ngaphansi, noma ekugxilweni okuphansi kwamaprotheni16,18,19.
Ukuhlolwa kwe-NT vial inversion (100 mg/mL) ku-pH 8, 7, 6 kanye no-154 mM NaCl (pH 8) ngemva kokufukamela ku-37°C.I-b NT CD spectra ene-154 mM NaF nangenayo ne-154 mM NaCl, ngokulandelanayo.I-Molar ellipticity ku-222 nm iguqulwa ibe ingxenye yokugoqa kwemvelo.c NT inversion assay (100 mg/mL) NT* (37 °C and 60 °C), NTA72R (37 °C), and His-NT-L6 (37 °C and 60 °C).d i-CD spectra ye-NT mutants NT*, NTA72R, kanye ne-His-NT-L6.I-Molar ellipticity ku-222 nm iguqulwa ibe ingxenye yokugoqa kwemvelo.e Ukuhlolwa kokuguqulwa kwe-NTFlSp, NTMiSp kanye ne-NTMiSp encishisiwe (100 mg/mL).Ibha yesikali 5 mm.f I-spectra ye-CD ye-NT, NTFlSp, NTMiSp kanye ne-NTMiSp encishisiwe.I-Molar ellipticity ku-222 nm iguqulwa ibe ingxenye yokugoqa kwemvelo.Ukubuka okugcwele kwe-NT ku-25 °C no-95 °C kuboniswa kuMfanekiso Ongezelelwe 8.
Ukugxiliswa kukasawoti womzimba kunquma ukusebenzisana kwe-electrostatic phakathi kwamayunithi amancane e-NT kanye nokuncipha kokudluliswa kwe-NT kuye ku-pH18 ephansi.Sithole ukuthi ubukhona be-154 mM NaCl kanye ne-NaF buye bavimbela i-gelation, ngokulandelanayo (Fig. 5a, b; I-Supplementary Fig. 2b) nokuthi lawa masawoti andise ukuzinza okushisayo kwe-NT monomers (Fig. 5b, Supplementary Fig. 8) .Iphinde iphakamise ukuthi ukuthuthukiswa kokuzinza, kunokuba i-dimerization, ivimbele ukwakheka kwejeli.
Ukuze siqhubeke sihlola indima ye-dimerization yamaprotheni nokuzinza ku-gelation, sisebenzise ama-mutant amabili, i-NT* ne-NTA72R, nayo ehlala i-monomeric ku-pH28.30 ephansi.I-NT* iwukushintshashintsha kokushaja okukabili lapho ukusatshalaliswa kweshaja ye-dipolela ebonakalayo ye-monomer kufiphaliswe, okuvimbela ukufiphala futhi kukhulisa kakhulu ukuzinza kwe-monomer.I-NTA72R iyi-dipole ekhokhisiwe, kodwa i-Arg-substituted Ala itholakala emngceleni we-dimer, ngakho ukuguqulwa kwezakhi zofuzo kuphazamisa ukuxhumana kweyunithi edingekayo ukuze kuncishiswe ukukhanya.Lapho i-incubation ku-37 ° C, i-NT * ayizange yakhe i-hydrogel, kuyilapho i-NTA72R yenza ijeli ye-opaque imizuzu engu-15 (Fig. 5c).Njengoba kokubili i-NT* ne-NTA72R ingakwazi ukufiphaza kodwa ihluke ekuzinzeni kwe-monomer (Fig. 5d), le miphumela iphakamisa ngokuqinile ukuthi ukuzinza okuphezulu kwe-thermodynamic kuvimbela i-NT ekugezeni.Lokhu futhi kusekelwa iqiniso lokuthi i-HT * yenza ijeli uma ingazinzile ekushiseni okuphezulu (ngemuva kwemizuzu engu-8 ku-60 ° C; Umdwebo 5c).Phambilini kubonisiwe ukuthi okuqukethwe okuphezulu kwe-methionine ku-NT kunyibilikisa ukugoqa kwayo kwemvelo nokuthi izisetshenziswa eziyisithupha ze-Met to Leu (okubhekiselwa kuzo lapha njenge-His-NT-L6) zizinza ngokuqinile i-monomer ye-NT46.Ngokusekelwe emcabangweni wokuthi ukuguquguquka kwesakhiwo kuyadingeka ekwakhekeni kwejeli le-NT, sithole ukuthi i-His-NT-L6 stable mutant ayizange igeli ku-37 °C (Figure 5c, d).Kodwa-ke, i-His-NT-L6 nayo yakha ijeli lapho ifakwa ku-60 ° С imizuzu engu-60 (Fig. 5c).
Ikhono le-NT lokuguqula libe izakhiwo ze-β-sheet futhi enze ama-hydrogel libonakala lisebenza kwezinye kodwa hhayi zonke izizinda ze-NT ze-spidroin.Ama-NT avela ezinhlotsheni zikasilika ezihlukene kanye nezinhlobo zezicabucabu, i-Trichonephila clavipes (NTFlSp), akha amajeli naphezu kokuqukethwe kwawo kwe-methionine ephansi kanye nokuzinza okuphezulu kokushisa (Umfanekiso 5e, f kanye neThebula Lokwengeza 2).Ngokuphambene, i-NT evela ku-ampullar protein spidroin encane evela ku-Araneus ventricosus (NTMiSp) ene-thermal stability ephansi kanye nokuqukethwe okuphezulu kwe-methionine akuzange kudale ama-hydrogel (Ithebula le-Supplementary 2 ne-Fig. 5e, f).Lokhu kokugcina kungase kuhlotshaniswe nokuba khona kwe-intramolecular disulfide bond29,47.Ngokuqhubekayo, lapho izibopho ze-disulfide ze-NTMiSp zincishiswa, zakha i-hydrogel ngemva kokufakwa ku-37 ° C imizuzu engu-10 (Fig. 5e).Sengiphetha, kufanele kuqashelwe ukuthi ukuguquguquka kwesakhiwo kubalulekile, kodwa hhayi kuphela, umbandela wokwakhiwa kwejeli evela ku-NT.Esinye isici esingase sibaluleke ukuthambekela kokwenza ama-amyloid fibrils, futhi ukuhlaziya nge-database ye-zipper kanye ne-algorithm ye-Waltz kubonise ukuhlobana phakathi kwekhono lokwakha ama-gel kanye nokuba khona kwezifunda ze-amyloidogenic, kanye nezinga lezifunda ezibikezelwe. ukwakha uziphu steric.Kube khona ukuhlobana (Ithebula Lokwengeza 2 kanye ne-Supplementary Fig. 9).
Ikhono le-NT lokwenza ama-fibril futhi enze amajeli ngaphansi kwezimo ezivumayo lisiholele ekucabangeni ukuthi ukuhlanganiswa kwe-NT nezinye izingcezu zamaprotheni kusengakha amajeli anomsebenzi ogcwele wozakwethu be-fusion.Ukuze sihlole lokhu, sethule iphrotheni ye-fluorescent eluhlaza (GFP) kanye ne-purine nucleoside phosphorylase (PNP) ku-C-terminus ye-NT, ngokulandelanayo.Amaprotheni ahlanganisiwe atholakala ku-E. coli enesivuno esiphezulu kakhulu sokugcina (150 mg/L kanye ne-256 mg/L shake flask cultures for His-NT-GFP and His-NT-PNP, ngokulandelana), ngokuhambisana nalokho okubonisiwe. kwamanye amaprotheni ahlanganiswe ku-NT Ref.30. I-His-NT-GFP (300mg / mL) kanye ne-His-NT-PNP (100mg / mL) amaprotheni e-fusion akha ama-gel ngemva kwamahora angu-2 namahora angu-6.5 ku-37 ° C futhi, okubalulekile, ingxenye ye-GFP yahlala ingashintshiwe.kuqashelwe ngemva kwe-gelation, ne-> 70% ye-fluorescence intensity yokuqala esele ngemva kwe-gelation (Fig. 6a).Ukuze silinganise umsebenzi we-PNP kuzisombululo namajeli akhe we-NT-PNP, kwakudingeka sihlambulule iphrotheni ye-fusion ne-NT ngoba umsebenzi we-enzymatic wokulungiselela okuhlanzekile wawungaphandle kwebanga lokutholwa kokuhlolwa ekugxilweni kwe-gelling.Ijeli elakhiwe ngengxube equkethe u-0.01 mg/mL I-His-NT-PNP kanye ne-100 mg/mL NT igcine u-65% womsebenzi wokuqala we-enzymatic wamasampuli afakwe ngaphambili (Fig. 6b).Ijeli yahlala iqinile ngesikhathi sokulinganisa (I-Supplementary Fig. 10).
ukuqina kwe-fluorescence ehlobene ngaphambi nangemuva kokugezwa kwe-His-NT-GFP (300 mg/mL) kanye nebhodlela ehlanekezelwe equkethe i-Hidrogel ye-His-NT-GFP (300 mg/mL) ngaphansi kokukhanya okubonakalayo ne-UV.Amaphuzu abonisa izilinganiso ngazinye (n = 3), amabha wamaphutha abonisa ukuchezuka okujwayelekile.Inani elimaphakathi liboniswa phakathi nendawo yamabha amaphutha.b Umsebenzi we-PNP watholwa ngokuhlaziywa kwe-fluorometric kusetshenziswa izixazululo namajeli ahlanganisa i-NT (100 mg/ml) kanye nengxube equkethe i-0.01 mg/ml yakhe-NT-PNP kanye ne-100 mg/ml Amadola amasha aseTaiwan.Okufakiwe kukhombisa ibhodlela elihlanekezelwe eliqukethe i-hydrogel equkethe i-His-NT-PNP (ibha yesikali esingu-5 mm).
Lapha, sibika ukwakheka kwama-hydrogel asuka ku-NT namanye amaprotheni e-spidroin aphinde ahlanganiswe ngokufukamela isisombululo samaprotheni ku-37°C (Umfanekiso 1).Sibonisa ukuthi i-gelation ihlotshaniswa nokuguqulwa kwe-α-helices ibe yizingqimba ze-β kanye nokwakhiwa kwama-fibrils afana ne-amyloid (Fig. 3 kanye ne-4).Lokhu okutholakele kuyamangaza njengoba ama-NTs eyi-globular eyi-five-helix eyinqwaba eyaziwa ngokuncibilika kwawo okuphezulu kakhulu kanye nokuzinza okuphezulu ekugxileni >200 mg/mL ku-4°C izinsuku ezimbalwa27.Ngaphezu kwalokho, ama-NT aphindaphindeka kalula ngemva kokukhishwa kokushisa ekugxilweni kwamaprotheni aphansi ku-µM.Ngokusho kwemiphumela yethu, ukwakheka kwe-fibril kudinga inhlanganisela> 10 mg / mL amaprotheni okuhlushwa kanye nokushisa okuphakeme kancane (Fig. 1).Lokhu kuhambisana nomqondo wokuthi ama-amyloid fibrils angakha kusuka kumaprotheni ahlanganiswe yi-globular asesimweni esingasombululeki kancane ngenxa yokuguquguquka kokushisa ngaphansi kwezimo ze-physiological 48.Izibonelo zamaprotheni angena kulokhu kuguqulwa zihlanganisa i-insulin49,50, β2-microglobulin, i-transthyretin ne-lysozyme51,52,53.Nakuba i-NT iyi-α-helix esimweni sayo sokuzalwa, cishe u-65% ​​we-polypeptide chain ihambisana nokwakheka kweziphu ezisteric (Fig. 4e) 45.Njengoba i-monomer i-dynamically mobile46, ingadalula lezi zifunda ze-amyloidogenic ezingaba khona emazingeni okushisa aphakeme ngokusesilinganisweni futhi ekugxilweni okuphezulu kwenani lamaprotheni angafinyelela ekugxilweni okubalulekile kwe-amyloid fibril formation54.Ukulandela lokhu kucabanga, sithole ukuhlobana okungekuhle phakathi kokuhlushwa kwe-spidroin nesikhathi se-gelation (Fig. 1c), futhi uma i-monomeric NT conformation iqiniswa noma ngokuguquguquka (NT*, His-NT-L6) noma ngokufaka usawoti, kungavimbela ukwakheka kwama-hydrogel (Fig. 5).
Ezimweni eziningi, ama-amyloid fibrils ayanyamalala esixazululweni njengemvula, kodwa ngaphansi kwezimo ezithile angenza ama-hydrogels55,56,57.Ama-fibril akha i-Hydrogel ngokuvamile ane-aspect ratio ephezulu futhi enza amanethiwekhi azinzile anezinhlangothi ezintathu ngokusebenzisa i-molecular entanglement,55,58 ehambisana nemiphumela yethu.Ngokwakhiwa kwe-hydrogel ku-vitro, amaprotheni avame ukuvezwa ngokugcwele noma kancane, isibonelo, ngokuchayeka ku-organic solvents, izinga lokushisa eliphezulu (70-90 ° C) kanye / noma i-pH ephansi (1.5-3.0) 59,60,61,62.Ama-spidroin hydrogel achazwe lapha awadingi ukucutshungulwa kanzima, futhi awadingi ama-agent axhumanisayo ukuze azinzise ama-hydrogel.
Kuke kwabikwa ngaphambilini ukuthi i-spidroin iyaphinda kanye nama-QD, abonakala eshintsha i-β-sheet ngesikhathi sokuphothwa kukasilika, enza ama-hydrogel.Uma kuqhathaniswa nalokho esikutholile, izikhathi zokufukamela kanye/noma amazinga okushisa ekufukamela ayemade kakhulu noma ngaphezulu, ngokulandelana, futhi ama-hydrogel avelayo ayevame uku-opaque (Umfanekiso 7 kanye Nethebula Lokwengeza 1) 37, 38, 63, 64, 65, 66, 67, 68 , 69. Ngaphezu kwezikhathi zejeli ezisheshayo, ama-hydrogel e-NT >300 mg/mL (30%) asebenza kahle kakhulu kunawo wonke amanye amaprotheni kasilika wesicabucabu achazwe kabusha, kanye nama-hydrogel emvelo njenge-gelatin, i-alginate (2%), i-agar (0.5 % ) kanye ne-collagen.(0.6%) (Umfanekiso 7 kanye Nezithasiselo Amathebula 1 kanye 3)37,39,66,67,68,69,70,71,72,73,74.
Isikhathi sejeli kanye ne-elastic modulus yama-hydrogel kulolu cwaningo kwaqhathaniswa namanye ama-hydrogel asekelwe ku-spidroin kanye nama-hydrogel emvelo akhethiwe.Izinkomba zinikezwa kanye nencazelo yezimo ze-gelation.I-APS Ammonium persulfate, izinga lokushisa legumbi.Idatha 37, 38, 39, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
Izicabucabu zibonakala zenze izindlela zokuvimbela i-spidroin ukuthi ingageli ngesikhathi sokugcinwa.Naphezu kokugcwala okuphezulu kwamaprotheni endlaleni kasilika, isifunda esikhulu sokuphindaphinda esihlotshaniswa nesizinda se-terminal sisho ukuthi ukugxila okubonakalayo kwe-NT ne-CT ku-gland kuhambisana cishe no-10-20 mg/ml, emngceleni walolu cwaningo.edingekayo ukuze kubonwe ukwakheka kwe-hydrogel ku-in vitro.Ngaphezu kwalokho, ukugxila okufanayo kosawoti 16 kuzinzile i-NT, njengasezindlala zesilika (Fig. 5b).Ukuhlanganiswa kwe-NT kuye kwacutshungulwa ku-E. coli cytosol futhi kwatholakala ukuthi igoqeke yaqina kakhulu kunalapho ihlolwa ku-in vitro, okubonisa futhi ukuthi usawoti noma ezinye izici zivimbela ukuhlangana kwawo ku-vivo.Kodwa-ke, ikhono le-NTs lokuguqula libe ama-β-sheet fibrils angase abaluleke ekwakhekeni kwe-filament futhi kufanele aphenywe ezifundweni ezizayo.
Ngaphezu kwezici zenoveli ze-NT-amyloid-like fibril kanye nokwakheka kwe-hydrogel okuphawulwe kulolu cwaningo, sibonisa futhi ukuthi lesi simo singase sibe nezinhlelo zokusebenza ze-biotechnological and biomedical (Fig. 8).Njengobufakazi bomqondo, sihlanganise i-NT ne-GFP noma i-PNP futhi sabonisa ukuthi iphrotheni ye-fusion iphinde yakha ama-hydrogel uma efakwe ku-37 ° C nokuthi ama-fractions e-GFP kanye ne-PNP agcina kakhulu umsebenzi wawo ngemva kokugcoba (Umfanekiso 6).I-Nucleoside phosphorylases ibalulekile ekusungulweni kwe-nucleoside analogues75, okwenza ukutholakala kwethu kuhambisane nemboni ye-biopharmaceutical.Umqondo wokuveza amaprotheni e-fusion akha ama-hydrogel asobala ngaphansi kwezimo ezivumayo uvumela ukwakhiwa kwama-hydrogel asebenzayo anezindawo ezivumayo zokusetshenziswa okubanzi okufana ne-enzyme immobilization, ukukhululwa kwezidakamizwa okulawulwayo kanye nobunjiniyela bezicubu.Ukwengeza, i-NT ne-NT* zingamaki enkulumo asebenza kahle30, okusho ukuthi i-NT nokuhluka kwayo kungasetshenziselwa ukukhiqizwa okuphezulu kwamaphrotheni ahlanganisiwe ancibilikayo nokudala okulandelayo kwamaprotheni aqondiwe angenakunyakaziswa kuma-hydrogel e-3D.
I-NT iyancibilika, i-α-helical futhi izinzile ekugxiliseni okuphansi (µM) no-37°C.Emazingeni okushisa afanayo, kodwa ekugxiliseni okukhulayo (>10 mg/ml), i-NT yenza amajeli ahlanganisa ama-amyloid-like fibrils.Amaprotheni e-NT fusion aphinde enze ama-fibrillar gels anezingcezu ze-fusion ezisebenza ngokugcwele, okuvumela amaprotheni ahlukahlukene ukuthi angakwazi ukuhamba kuma-hydrogel e-3D esebenzisa i-NT.Ngezansi: NT (PDB: 4FBS) nemifanekiso yamanethiwekhi efayibha kanye nezakhiwo zamaprotheni ezihlotshaniswayo (okucatshangwayo futhi okungadwetshwe esikalini, i-GFP PDB: 2B3Q, 10.2210/pdb2B3Q/pdb; PNP PDB: 4RJ2, 10.2210/pdb4bJ2).
Abakhi (bona Ithebula Le-Supplementary 4 ukuze uthole uhlu oluphelele oluhlanganisa ukulandelana kwe-amino acid) kwenziwa i-plasmid pT7 futhi yaguqulwa yaba yi-E. coli BL21 (DE3).I-E. coli equkethe ama-plasmid aklanywe ngonjiniyela yajovwa emhluzini we-Luria ohlanganiswe ne-kanamycin (70 mg/l) futhi yakhuliswa ngobusuku obungu-30°C no-250 rpm.Isiko labe seligonywa ngo-1/100 ku-LB medium equkethe i-kanamycin futhi yakhuliswa ku-30°C no-110 rpm kuze kube yilapho i-OD600 ifinyelela ku-0.8.Ocwaningweni lwe-NMR, amagciwane atshalwa ku-M9 okumaphakathi okuqukethe u-2 g we-D-glucose 13C (Aldrich) kanye ne-1 g ye-ammonium chloride 15N (Cambridge Isotope Laboratories, Inc.) ukuze kufakwe ilebula yamaprotheni ngama-isotopu.Yehlisa izinga lokushisa libe ngu-20 degrees Celsius futhi wenze ukubonakaliswa kwamaprotheni ngo-0.15 mM isopropylthiogalactopyranoside (ukugxila kokugcina).Ngemuva kokubonakaliswa kwamaprotheni ebusuku, amaseli avunwa ku-7278×g, 4 ° C imizuzu engu-20.Ama-cell pellets aphinde amiswa ku-20 mM Tris-HCl, pH 8, futhi aqandiswa kuze kube yilapho esetshenziswa futhi.Amaseli ancibilikisiwe ahlanjululwa kusetshenziswa isiphazamisi seseli (imishini yochungechunge lwe-TS, i-Constant Systems Limited, e-England) ngo-30 kPa.Khona-ke ama-lysates afakwa i-centrifuged ku-25,000 g imizuzu engu-30 ku-4 ° C.Ku-NTMiSp, i-pellet yabe isiphinde yamiswa ku-2 M urea, 20 mM Tris-HCl, pH 8, futhi yenziwa i-sonication imizuzu engu-2 (2 s on/off, 65%), bese i-centrifured futhi ku-25,000 xg, 4° C. ngaphakathi 30 imiz.I-supernatant yalayishwa kukholamu ye-Ni-NTA, yagezwa ngo-20 mM Tris-HCl, 2 mM imidazole, pH 8, futhi ekugcineni iphrotheni yakhishwa ngo-20 mM Tris-HCl, 200 mM imidazole, pH 8. Ukukhiqiza i-NT2RepCT kanye I-NTCT, ukugaya kwe-thrombin kwethula isayithi (i-ThrCleav) phakathi kwe-His ne-NT.Izindawo ze-Thrombin cleavage zikhona futhi ku-His-NT-ThrCleav-2Rep (ikhiqiza i-2Rep), i-His-thioredoxin-ThrCleav-NT (ikhiqiza i-NT), i-His-thioredoxin-ThrCleav-CT (ikhiqiza i-CT), i-His-Thioredoxin-ThrCleav-NT .* (ikhiqiza i-NT*), i-His-Thioredoxin-ThrCleav-NTA72R (ikhiqiza i-NTA72R), i-His-Thioredoxin-ThrCleav-NTFlSp (ikhiqiza i-NTF1Sp), kanye ne-His-Sulphur Redoxin-ThrCleav-NTMiSp (ikhiqiza i-NTMiSp).Izakhiwo zigaywe nge-thrombin (1:1000) futhi zahlanjululwa ngobusuku ku-4° C. nge-20 mM Tris-HCl, pH 8, kusetshenziswa ulwelwesi lwe-Spectra/Por dialysis olunomkhawulo wesisindo se-molecular of 6-8 kDa.Ngemva kokukhishwa kwe-dialysis, isixazululo silayishwa kukholamu ye-Ni-NTA futhi ukungcola okuqukethe amaprotheni anentshisekelo kuyaqoqwa.Ukugxila kwamaprotheni kwanqunywa ngokulinganisa ukumunca kwe-UV ku-280 nm kusetshenziswa i-coefficient yokushabalala yephrotheni ngayinye, ngaphandle kwe-NTF1Sp, esebenzise ukuhlolwa kwe-Bradford ngokuya ngephrothokholi yomkhiqizi.Ubumsulwa banqunywa i-SDS polyacrylamide (4–20%) ijeli electrophoresis kanye ne-Coomassie brilliant blue staining.Amaprotheni ayegxilwe kusetshenziswa izihlungi ze-centrifuge (i-VivaSpin 20, i-GE Healthcare) ku-4000 xg ne-10 kDa isisindo se-molecular cutoff emijikelezweni yemizuzu engu-20.
Ncibilikisa isixazululo sephrotheni bese uphayiphi ngokucophelela u-150 µl kubhodlela le-septum elicacile elingu-1 ml (8 x 40 mm Thermo Scientific).Amashubhu ayevalwa futhi avalwa nge-parafilm ukuvimbela ukuhwamuka.Amasampula (n = 3) afakwe ku-37°C noma ku-60°C futhi ngezikhathi ezithile aguqulwa ukuze abheke ukugeleza kwegazi.Amasampula angazange afakwe ijeli afukanyelwa okungenani isonto elilodwa.Yehlisa amabhondi e-NTMiSp disulfide ngo-10 mM DTT ngeprotheni engu-10 µM.Ukuhlaziya i-gelation yezingubo zikasilika wesicabucabu semvelo, isicabucabu sebhuloho saseSweden sasikwa, izindlala ezimbili eziyinhloko ezikhuphukile zafakwa ku-200 μl we-20 mM Tris-HCl buffer pH 8 futhi zasikwa ukuze kuvunyelwe ukunamathela kwehlukana nezindlala..Okuqukethwe kwezindlala kuncibilika ku-buffer, 50 µl ukuze kutholwe isisindo esomile (ngokufukamela izitsha ezivulekile ku-60 °C kuya esisindweni esingaguquki) kanye no-150 µl we-gelation ku-37 °C.
Ijiyomethri/ithuluzi lokulinganisa lenziwe ngensimbi engagqwali kusetshenziswa ipuleti elihambisanayo elinobubanzi obungaphezulu buka-20 mm kanye negebe elingu-0.5 mm.Shisisa isampula ukusuka ku-25 °C ukuya ku-45 °C futhi ubuyele ku-25 °C ngenani elingu-1 °C ngomzuzu usebenzisa ipuleti le-Peltier eliphansi lensimbi engagqwali.Izilinganiso zokudlidliza zenziwa imvamisa engu-0.1 Hz futhi endaweni eqondile ye-viscoelastic yezinto ngobunzima obungu-5% no-0.5% kumasampuli we-100 mg/mL kanye no-300–500 mg/mL, ngokulandelana.Sebenzisa ikamelo lokuswakama ngokwezifiso ukuze uvimbele ukuhwamuka.Idatha yahlaziywa kusetshenziswa i-Prism 9.
Ukuqoqa i-spectra ye-infrared (IR) ekamelweni lokushisa kusuka ku-800 kuya ku-3900 cm-1.Idivayisi ye-ATR, kanye nendlela yokukhanya nge-spectrometer, ihlanzwa ngomoya owomile ohlungiwe ngaphambi nangesikhathi sokuhlolwa.Izixazululo (500 mg/mL ukuze kuncishiswe iziqongo zokumunca amanzi ku-spectra) zafakwa ngamapayipi kumakristalu, futhi amajeli (500 mg/mL) akhiwa ngaphambi kokulinganisa abese edluliselwa kumakristalu (n = 3).Izikena eziyi-1000 zarekhodwa ngokulungiswa okungu-2 cm-1 kanye nomjikelezo onguziro we-2. Okuphuma kokunye kubalwe kusetshenziswa i-OPUS (Bruker) kusetshenziswa ububanzi obushelelezi bamaphuzu ayisishiyagalolunye.I-spectra yayijwayele ukuya endaweni efanayo yokuhlanganisa phakathi kuka-1720 no-1580 cm-1 kusetshenziswa i-F. Menges "Spectragryph - Optical Spectroscopy Software".Ku-spectroscopy ye-ATR-IR, ukujula kokungena kwe-infrared beam kusampula kuncike kwinombolo ye-wave, okuholela ekumunceleni okunamandla kuzinombolo zamagagasi aphansi kunasezinombolo zamagagasi aphezulu.Le miphumela ayizange ilungiswe ku-spectra eboniswe ku-Fig.3 ngoba mancane kakhulu (Supplementary Fig. 4).I-spectra elungisiwe yalesi sibalo ibalwe kusetshenziswa isofthiwe ye-Bruker OPUS.
Empeleni, ukulinganisa okuphelele kokuhlanganiswa kwamaprotheni kungenzeka ngemva kokuhlukaniswa okuthembekile kwezingxenye ngaphakathi kwe-amide I peak.Nokho, ezinye izithiyo ziyaphakama ekusebenzeni.Umsindo ku-spectrum ungavela njengeziqongo (ezingemanga) phakathi ne-deconvolution.Ngaphezu kwalokho, inani eliphakeme ngenxa yokugoba kwamanzi liqondana nendawo ye-amide I peak futhi lingase libe nobukhulu obufanayo bamasampuli aqukethe inani elikhulu lamanzi, njengejeli enamanzi efundwa lapha.Ngakho-ke, asizange sizame ukubola ngokuphelele i-amide I peak, futhi ukuqaphela kwethu kufanele kucatshangelwe kuphela ekusekeleni ezinye izindlela ezifana ne-NMR spectroscopy.
Izixazululo ze-50 mg/ml NT kanye ne-His-NT2RepCT zenziwa ebusuku ku-37°C.I-hydrogel yabe isihlanjululwa ngo-20 mM Tris-HCl (pH 8) yaya ekuhlanganiseni okungu-12.5 mg/ml, yanyakaziswa kahle futhi yafakwa ngamapayipi ukuze kuphuke ijeli.Okulandelayo, i-hydrogel yahlanjululwa izikhathi ezingu-10 nge-20 mM Tris-HCl (pH 8), u-5 μl wesampula wasetshenziswa kugridi yethusi embozwe nge-formvar, futhi isampula eyengeziwe yakhishwa ngephepha lokusula.Amasampuli ahlanzwa kabili ngo-5 µl wamanzi we-MilliQ futhi angcoliswa nge-uranyl formate engu-1% imizuzu emi-5.Susa ibala elidlulele ngephepha elimuncayo, bese womisa nge-mesh emoyeni.Ukuthwebula izithombe kwenziwa kulawa magridi kusetshenziswa i-FEI Tecnai 12 Spirit BioTWIN esebenza ngo-100 kV.Izithombe zirekhodwe ku-x 26,500 kanye no-x 43,000 ekukhulisweni kusetshenziswa ikhamera ye-Veleta 2k × 2k CCD (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).Kusampula ngayinye (n = 1), izithombe eziyi-10–15 zarekhodwa.I-ImageJ (https://imagej.nih.gov/) isetshenziselwe ukuhlaziya isithombe kanye nokukalwa kwamadiamitha efayibha (n = 100, imicu ehlukene).I-Prism 9 yasetshenziswa ukwenza izivivinyo zika-t ezingabhangqiwe (ezinemisila emibili).Isilinganiso se-His-NT2RepCT ne-NT fibrils bekuyi-11.43 (SD 2.035) kanye ne-7.67 (SD 1.389) nm, ngokulandelanayo.Isikhathi sokuzethemba (95%) singu-4.246 kuya ku-3.275.amadigri enkululeko = 198, p <0.0001.
U-80 µl wamasampula oketshezi aqukethe u-10 µM thioflavin T (ThT) akalwe ngama-triplicate (n = 3) ngaphansi kwezimo ezimile kusetshenziswa amapuleti aphansi acacile angama-Corning 96-amnyama ngezansi (Corning Glass 3881, USA).Umehluko we-Fluorescence urekhodwe kusetshenziswa isihlungi se-excitation esingu-440 nm kanye nesihlungi se-480 nm emission (i-FLUOStar Galaxy evela ku-BMG Labtech, Offenburg, Germany).Isignali ye-ThT ayizange igcwale noma icinywe, njengoba ukuhlolwa okunezingqikithi ezahlukene ze-ThT kwenziwa ngaphandle kokushintsha ukuqina kwesignali.Rekhoda ukumunca ku-360 nm ukuze uthole ukulinganisa kwenkungu.Ocwaningweni lwembewu, amajeli angu-100 mg/mL akhiwe ku-37° C., amiswa kabusha, futhi asetshenziselwe ukuhlwanyela ngezilinganiso ze-molar zika-5%, 10%, kanye no-20%.Idatha yahlaziywa kusetshenziswa i-Prism 9.
Ncibilikisa izitoko ze-His-NT2RepCT ne-NT >100 mg/mL eqhweni bese uhlunga ngesihlungi esingu-0.22 µm.Ukugxila kubalwe ngokulinganisa ukumunca ku-280 nm kusetshenziswa i-Nanodrop.Emithonjeni yepuleti elingabopheli elimnyama elingu-96 (i-Corning) elinephansi elicacile, amasampuli ahlanjululwe abe ngu-20 mg/ml ku-20 mM Tris-HCl pH 8 futhi axutshwa no-5 μM ThT (ukugxiliswa kokugcina), ukugxiliswa kwesampula okuphelele 50 μl umthamo.Amasampuli athathwa njalo emizuzwini eyi-10 ku-37 ° C kusibonakhulu se-CellObserver (Zeiss) esinesiteshi sokukhanya esidluliswayo kanye namasethi okuhlunga e-FITC kanye ne-emission ye-ThT imaging.Ilensi engu-20x/0.4 isetshenziselwa ukuthwebula izithombe.I-Zen Blue (Zeiss) kanye ne-ImageJ (https://imagej.nih.gov/) zisetshenziselwe ukuhlaziya isithombe.Amajeli aphinde alungiswa kusuka ku-NT kanye nezixazululo ze-His-NT2RepCT ekuhlanganiseni okungu-50 mg/mL okuqukethe u-20 mM Tris pH 8 kanye no-5 µM ThT futhi efakwe ku-37°C imizuzu engu-90.Izingcezu zejeli zadluliselwa emthonjeni omusha oqukethe u-20 mM Tris, pH 8, kanye no-5 μM ThT epuleti eliphansi elimnyama elingabopheli elingu-96 elicace kahle.Thola i-fluorescence eluhlaza kanye nezithombe zensimu ekhanyayo ekukhuliseni okungu-20x/0.4.I-ImageJ isetshenziselwe ukuhlaziya isithombe.
Isixazululo se-NMR spectra sitholwe ku-310 K ku-spectrometer ye-Bruker Avance Neo engu-600 MHz efakwe i-QCI Quadrupole Resonance Pulsed Gradient Field Cryoprobe (HFCN).Amasampula e-NMR aqukethe u-10 mg/mL wephrotheni ehambisanayo enelebuli engu-13C, 15N, encibilike ngo-20 mM Tris-HCl (pH 8), 0.02% (w/v) NaN3, 5% DO (v/v), (n = 1) .Ukushintsha kwamakhemikhali kwe-NT2RepCT ku-pH 6.7 kusetshenziswe ukunikeza i-peak 23 ku-spectrum ye-2D ye-15N-HSQC.
I-magic angle spinning solid NMR (MAS) spectra ye-13C, 15N-anelebula ama-hydrogel aqoshwe ku-spectrometer ye-Bruker Avance III HD ku-800 MHz efakwe i-3.2 mm 13C/15N{1H} i-electronless probe.Isampula lokushisa lalilawulwa kusetshenziswa izinga lokushisa eliguquguqukayo lomfudlana wegesi ku-277 K. I-dimensional dipole rotational resonance (DARR)76 kanye ne-radio frequency reconnection (RFDR)77 spectra itholwe kumafrikhwensi we-MAS we-12.5 kHz no-20 kHz, ngokulandelanayo.I-Cross polarization (CP) isuka ku-1H iye ku-13C yenziwe kusetshenziswa irempu ewumugqa ukusuka ku-60.0 ukuya ku-48.0 kHz ku-1H, 61.3/71.6 kHz ku-13C (ku-12.5/20 kHz MAS) nesikhathi sokuxhumana esingu-0.5–1 ms.I-Spinal6478 decoupling ku-73.5 kHz isetshenziswe phakathi nokuqoqwa kwedatha.Isikhathi sokutholwa sasingu-10 milliseconds futhi ukubambezeleka komjikelezo kwakuyimizuzwana engu-2.5.Ukuhlobana okuxhunywe okukodwa kwe-Cα/Cβ okubonwe ku-spectra ye-RFDR kwabelwa ngokusekelwe kushintsho lwamakhemikhali ohlobo lwezinsalela kanye nokuxhumana okuxhunywe ngokuphindaphinda ku-spectra ye-DARR.
Isizindalwazi se-Zipper79 (https://services.mbi.ucla.edu/zipperdb/) sisetshenziswe ukuhlola ukuthambekela kwe-flutter namandla e-Rosetta ye-NT, NTFlSp, kanye ne-NTMiSp.Isizindalwazi se-Zipper sihlanganisa i-Rosetta Energy80, ehlanganisa imisebenzi embalwa yamahhala yamandla ukuze imodeli nokuhlaziya ukwakheka kwamaprotheni.Izinga lamandla -23 kcal/mol noma ngaphansi libonisa ukuthambekela okuphezulu kwe-fibrillate.Amandla aphansi asho ukuzinza okwengeziwe kwama-β-strands amabili ekuhlanganiseni uziphu.Ngaphezu kwalokho, i-algorithm ye-Waltz yasetshenziselwa ukubikezela izifunda ze-amyloidogenic ku-NT, NTFlSp kanye ne-NTMiSp Ref.81. (https://waltz.switchlab.org/).
Isixazululo sephrotheni ye-NT sixutshwe ne-2-(N-morpholino)ethanesulfonic acid (MES) buffer ku-pH 5.5 kanye no-6.0 ukuze kwehliswe i-pH ku-pH 6 no-7, ngokulandelanayo.Ukugxila kokugcina kwamaprotheni kwaba ngu-100 mg/ml.
Izilinganiso zenziwa ku-J-1500 CD spectrometer (JASCO, USA) kusetshenziswa i-cuvette engu-300 μL enendlela yokubona engu-0.1 cm.Amaprotheni ahlanjululwe ku-10 μM (n = 1) ku-20 mM ye-phosphate buffer (pH 8).Ukuhlaziya ukuzinza kwamaprotheni lapho kukhona usawoti, amaprotheni ahlaziywa ekugxilweni okufanayo (n = 1) ku-20 mM phosphate buffer (pH 8) equkethe i-154 mM NaF noma i-NaCl, ngokulandelanayo.Izikena zezinga lokushisa zirekhodwe ku-222 nm ukusuka ku-25°C kuya ku-95°C nezinga lokushisa elingu-1°C/min.Ingxenye yamaprotheni agoqwe ngokomdabu yabalwa kusetshenziswa ifomula (KDmeasure – KDfinal)/(KDstart – KDfinal).Ngaphezu kwalokho, ama-spectra amahlanu aqoshwa isampula ngayinye ukusuka ku-260 nm kuya ku-190 nm ku-25°C futhi ngemva kokushisisa kuya ku-95°C.Izibukeli ezinhlanu zilinganiselwe, zashelelwa futhi zaguqulwa zaba yi-molar ellipticity.Idatha yahlaziywa kusetshenziswa i-Prism 9.
Amandla e-fluorescence e-His-NT-GFP (300 mg/mL, 80 µL) akalwe nge-triplicate (n = 3) kumapuleti angama-96-well Corning anephansi elibonisa ngale emnyama (Corning Glass 3881, USA) ngaphansi kwezimo ezimile.Linganisa amasampula ngesifundi sepuleti esisekelwe ku-fluorescence esinobude begagasi obujabulisayo obungu-395 nm bese urekhoda ukukhishwa ku-509 nm ngaphambi kwe-gelation namahora angu-2 kamuva ku-37°C.Idatha yahlaziywa nge-Prism 9.
Ikhithi yokuhlola umsebenzi we-Purine nucleoside phosphorylase (indlela ye-fluorometric, i-Sigma Aldrich) isetshenziswe ngokulandela imiyalelo yomkhiqizi.Ukuze ulinganise umsebenzi kumajeli nezisombululo eziqukethe i-His-NT-PNP, hlanganisa u-10 ng we-His-NT-PNP no-100 mg/mL NT kuvolumu ephelele engu-2 µL ngoba ijeli inikeze isignali ngaphezu kwesikhawu sokutholwa sesethi.Izilawuli zamajeli nezisombululo ngaphandle kwe-His-NT-PNP zifakiwe.Izilinganiso zenziwe kabili (n = 2).Ngemva kokuba umsebenzi ulinganisiwe, ingxube yokusabela isusiwe futhi ijeli yathwetshulwa ukuze kuqinisekiswe ukuthi ijeli lihlala linjalo ngesikhathi sokulinganisa.Idatha yahlaziywa kusetshenziswa i-Prism 9.
Ukuze uthole ulwazi olwengeziwe mayelana nesakhiwo sokutadisha, bheka i-abstract yocwaningo lwezeMvelo exhunywe kulesi sihloko.
Umfanekiso 1 no-2 wethula idatha yokuqala.1c, 2a–c, 3a, b, e–g, 4, 5b, d, f, kanye no-6, Amakhiwane Esengezo.3, umkhiwane owengeziwe.5a, d, umkhiwane ongeziwe.6 kanye nomkhiwane owengeziwe.8. Idatha Yedatha evela kulolu cwaningo isingathwe kusizindalwazi se-Zenodo https://doi.org/10.5281/zenodo.6683653.Idatha ye-NMR etholwe kulolu cwaningo ithunyelwe endaweni yokugcina ye-BMRBig ngaphansi kwe-ID yokufaka bmrbig36.Izakhiwo ze-GFP ne-PNP zithathwe ku-PDB (GFP 2B3Q, PNP 4RJ2).
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