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Inkcazo
310 10 * 1mm intsimbi engatyiwayo ebhijelwe ababoneleli ngeetyhubhu
| IBanga | 301 ,304 ,304L ,316 ,316L ,309 S, 310 ,321 |
| Umgangatho | ASTM A240, JIS G4304, G4305, GB/T 4237, GB/T 8165, BS 1449, DIN17460, DIN 17441 |
| Ukutyeba | 0.2-10.0mm |
| Ububanzi | 600mm min |
| Ubude | 2000mm-8000mm okanye njengesicelo sabathengi |
| Ukugqitywa komphezulu | NO1, No.4,2B, BA, 6K, 8K, Hair Line kunye PVC |
Ukuqulunqwa kwemichiza
| IBanga | C | Si | Mn | P≤ | S≤ | Cr | Mo | Ni | Okunye |
| 301 | ≤0.15 | ≤1.00 | ≤2.00 | 0.045 | 0.03 | 16-18 | - | 6.0 | - |
| 304 | ≤0.07 | ≤1.00 | ≤2.00 | 0.035 | 0.03 | 17-19 | - | 8.0 | - |
| 304L | ≤0.075 | ≤1.00 | ≤2.00 | 0.045 | 0.03 | 17-19 | - | 8.0 | |
| 309S | ≤0.08 | ≤1.00 | ≤2.00 | 0.045 | 0.03 | 22-24 | - | 12.0 | - |
| 310 | ≤0.08 | ≤1.5 | ≤2.00 | 0.045 | 0.03 | 24-26 | - | 19.0 | - |
| 316 | ≤0.08 | ≤1.00 | ≤2.00 | 0.045 | 0.03 | 16-18.5 | 2 | 10.0 | - |
| 316L | ≤0.03 | ≤1.00 | ≤2.00 | 0.045 | 0.03 | 16-18 | 2 | 10.0 | - |
| 321 | ≤0.12 | ≤1.00 | ≤2.00 | 0.045 | 0.03 | 17-19 | - | 9.0 | Ti≥5×C |
IiPropati zoomatshini
| IBanga | YS(Mpa) ≥ | TS (Mpa) ≥ | El (%) ≥ | Ukuqina(HV) ≤ |
| 301 | 200 | 520 | 40 | 180 |
| 304 | 200 | 520 | 50 | 165-175 |
| 304L | 175 | 480 | 50 | 180 |
| 309S | 200 | 520 | 40 | 180 |
| 310 | 200 | 520 | 40 | 180 |
| 316 | 200 | 520 | 50 | 180 |
| 316L | 200 | 480 | 50 | 180 |
| 321 | 200 | 520 | 40 | 180 |
Iiproteni zesilika yesigcawu (iiproteni zesilika yesigcawu) zinezicelo ezininzi ezinokubakho kuphuhliso lwebhayomatariya entsha, kodwa imo yazo emininzi kunye nendalo elungeleleneyo yokudibanisa izenza kube nzima ukuzifumana kwaye kulula ukuzisebenzisa.Apha sixela ukuba iiproteni ze-spidroin ezincinci eziphinda ziphinde ziphinde ziphinde ziphinde, ngokubalulekileyo, isizinda se-N-terminal (NT) ngokwayo ngokukhawuleza senza ii-hydrogel ezizixhasayo kunye ne-transparent kwi-37 °C.iiprotheyini ezidityanisiweyo ezibandakanya i-NT kunye neprotheni ye-fluorescent eluhlaza okanye i-purine nucleoside phosphorylase yenza iiprotheni ezidibeneyo ezisebenza ngokupheleleyo.Ii-Hydrogels.Iziphumo zethu zibonisa ukuba i-NT ephindaphindayo kunye neeprotheyini ezidibeneyo zibonelela ngemveliso ephezulu yokubonisa kwaye inika ii-hydrogels ezineempawu ezikhangayo ezifana nokungafihli, i-gelation ngaphandle kokudibanisa, kunye nokunyanzeliswa ngokuthe ngqo kweeprotheyini ezisebenzayo kuxinaniso oluphezulu.
Izigcawu zineenxalenye ezisixhenxe ezahlukeneyo zamadlala esilika, ngalinye livelisa uhlobo oluthile lwesilika.Zonke iintlobo ezisixhenxe zesilika zenziwe ngeeproteni zesigcawu zesilika (spidroins) malunga neentsalela ezingama-6000 ubude kwaye ziqulethe ummandla omkhulu wokuphinda osembindini ongqongwe yimimandla engqukuva ye-N- kunye ne-C-terminal (NT kunye ne-CT)1,2.Olona hlobo lwesilika oluhlolisiswa kakhulu, i-primary ampulla, iveliswa lidlala eliyiprimary ampulla.Kweli dlala, i-monolayer yeeseli ze-epithelial idibanisa iiprotheni ze-spidroin kwaye zifihla kwi-lumen yedlala, apho zikhoyo kwifom enyibilikayo (i-doping) ekugxininiseni okuphezulu kakhulu (30-50% w / v)3,4.Umbutho kunye nokulungelelaniswa kweeprotheyini eziphambili ze-ampullar spidroin kwi-gland kuye kwaxoxwa ngayo, kodwa ubungqina obuninzi bokuvavanya bubonisa ubukho be-helical ngokubanzi kunye / okanye i-random helical conformation kunye ne-micellar okanye i-lamellar structures5,6,7,8,9,10.Ngelixa iindawo eziphindaphindiweyo zilawula iimpawu ze-mechanical ze-silk fibers, ukwenza i-β-sheet nanocrystals kunye nezakhiwo ze-amorphous11,12,13,14,15, iindawo zokuphela zilawula iintambo ze-silk ekuphenduleni iimeko eziguqukayo kunye ne-silk gland16,17,18.Ngokulawula ukubunjwa kwe-silk, i-19. Imimandla ye-terminal igcinwa ngokuguquguqukayo kwaye umsebenzi wabo unokuba yinto eqhelekileyo kuzo zonke iiprotheni ze-spidroin 2,20,21.Ngexesha lokudlula kwidlala, i-pH ye-spidroin iyancipha ukusuka malunga ne-7.6 ukuya ku-<5.716 kwaye iyanda ngokucheba kunye nokwelula okuyanyaniswa yintshukumo ngomjelo ocutheka kancinci kancinci.Kwisisombululo, i-CT yi-α-helical constitutive parallel dimer17, kodwa ekuphenduleni i-pH ephantsi kunye nemikhosi ye-shear, i-CT ityhila kwaye itshintshe i-β-layers16, i-17, mhlawumbi ibangele i-β-layers kwimimandla ephindaphindiweyo yokuguqula i-16. I-NT i-monomeric phantsi kwe-monomeric phantsi kwe-16. iimeko ezibonisa iimeko kwilumen yedlala kunye nokulamla ukunyibilika kwe-spidroin, kodwa kwi-pH encitshisiweyo, ukuveliswa kwenani lamatyathanga asecaleni e-carboxylic acid kukhokelela ekudimeni kwe-NT ngepKa emalunga ne-6.5, ngaloo ndlela kuzinzisa i-NT kunye nokulungisa i-spidroin enkulu. izixa.uthungelwano16,18.Ngaloo ndlela, i-NT idlala indima ephambili ekubunjweni kwe-filament, ukutshintsha ukusuka kwi-monomer kwi-coating ukuya kwi-dimer kwi-fiber23,24,25.I-NT ihlala i-soluble kakhulu kwaye i-helical phantsi kwazo zonke iimeko ezifundwe ukuza kuthi ga ngoku16, 18, 19, 20, 26, 27, 28, 29, eyaphefumlela uphuhliso lwayo njengeleyibhile yokuphucula ukunyibilika kokuveliswa kweeproteni ezi-heterologous.
Iprotheyini yesilika yesigcawu esiphinda-phindayo, equka i-NT enye, ingingqi enye emfutshane, iCT enye, kunye nethegi ye-His6 (His-NT2RepCT) yokucoca, inyibilika kwisithinteli esimanzi njengeprotein yesigcawu sesilika kwaye ilinganisa iimpawu ezibalulekileyo zesigcawu sesilika. .ingxelo 25.31.I-His-NT2RepCT inokuphonswa kwiifiber eziqhubekayo usebenzisa umatshini we-biomimetic apho i-pH 8 i-soluble coating ikhutshelwa kwi-pH 525,32,33,34,35 yokuhlamba amanzi.I-Bioreactor fermentation ye-E. coli echaza i-His-NT2RepCT kunye ne-post-post-treatment elandelayo ibangele> i-14 g / L isivuno emva kokucocwa.Isivuno esiphezulu, ukunyibilika okuphezulu, kunye nokuphendula okwaneleyo kwe-His-NT2RepCT kwiimeko ze-acidic zonke zibalelwa kwi-NT23, 25, 34.
Apha sinika ingxelo ngokwenziwa ngokukhawuleza kwee-hydrogels ezicacileyo ezivela kwi-recombinant spidroin proteins, kuquka i-NT iyodwa, ngokufukamela isisombululo seprotheni kwi-37 °C.Ukusebenzisa i-thioflavin T fluorescence (ThT), iFourier iguqula i-infrared spectroscopy (FTIR), i-nuclear magnetic resonance spectroscopy (NMR) kunye ne-electron microscopy yothumelo (TEM), sifumanise ukuba i-NT kunye ne-microspider proteins zingena kwi-structural transformation ibe yi-β-sheets kunye ne-amyloid-like fibrils. xa iijeli zenziwe.Ukongezelela, iiprotheyini ezixutywayo ze-NT kunye neprotheni ye-fluorescent eluhlaza (GFP) okanye i-purine nucleoside phosphorylase (PNP) yenza i-hydrogels eneziqhekeza ezidibeneyo ezisebenza ngokupheleleyo.Ukubonakaliswa komgangatho ophezulu kwimikhosi ye-heterologous, kunye nokuqulunqwa ngokukhawuleza kwee-hydrogels phantsi kweemeko ze-physiological, kuvula ithuba lokuvelisa i-hydrogels enexabiso eliphantsi kunye nemisebenzi yobunjineli.
Ngokungafaniyo neeprotheni ezininzi ze-spidroin ezixeliweyo36, i-His-NT2RepCT izinzile kwi-Tris-HCl buffer kwi-pH 8 kwaye inokugxininiswa ukuya kwi-500 mg / mL ngaphandle kwemvula25.Ngoko ke, samangaliswa ukufumanisa ukuba le proteni yenza ngokukhawuleza i-optical clear, i-self-supporting hydrogels xa ifakwe kwi-37 ° C (Fig. 1b-d).Uphononongo olongezelelweyo lubonise ukuba i-His-NT2RepCT gelation yenzeke phezu koluhlu olubanzi lweeprotheni (10-300 mg / mL) kwaye ukuba le ngqungquthela yayichasana ngokungafaniyo kunye nexesha le-gelation (umzobo 1c kunye ne-Supplementary Fig. 1).Ukufumanisa ukuba zeziphi iinxalenye ze-Hi-NT2RepCT mediate hydrogel formation, emva koko sihlolisise i-domain nganye ngabanye kunye neendibaniselwano ezahlukeneyo zisebenzisa i-flask inversion assay (Umfanekiso 1a, b).Zonke iinqununu ezivavanyiweyo ze-spidroin ezihlaziyiweyo zenze iigels (kwi-protein concentration ye-300 mg / mL) ngaphantsi kwe-1 h, ngaphandle kwe-2Rep (umzobo 1b).Oku kuphakamisa ukuba i-NT kunye ne-CT yodwa, ngokudibeneyo, okanye ehambelana nokuphindaphinda, iyakwazi i-gel kwi-37 ° C kwaye i-tag ye-His6 ayichaphazeli le nkqubo kunoma yimuphi umlinganiselo obalulekileyo.Ukunika ingcamango eqhelekileyo yokuba i-NT yiprotheyini enyibilikayo kakhulu kwaye izinzile, kwaye iingxelo zangaphambili ze-spidroin hydrogels ezihlangeneyo zibalele iziphumo ze-gelation kwiinguqu ezihambelanayo kwiindawo eziphindaphindiweyo kunye / okanye ii-CTs, i-NT ngokwayo inokuba nayo.Ukufunyanwa kwegelation kwakungalindelekanga.Itheyibhile eyoNgezelelweyo 1) 37, 38, 39. Ngokuphawulekayo, i-NT sele isele i-gelled ngaphakathi kwemizuzu eyi-10 ekugxininiseni kwe-≥ 300 mg / mL (Umfanekiso 1c).Iimvavanyo ze-Vial inversion kunye neentlobo ezahlukeneyo ze-NT zibonise ukuba kwi> 50 mg / mL isisombululo se-NT sifakwe ngokukhawuleza kune-His-NT2RepCT kwi-concentration ehambelanayo (w / v, Figure 1c).
Ukumelwa okucwangcisiweyo kwezinto ezahlukeneyo zespidroin ezifundwe kulo msebenzi.b Ixesha lejeli kuma-37 °C kwiiprotheni ezahlukeneyo ze-spidroin (300 mg/mL) ziqinisekiswe ngokujikwa kwevial.I-CT gel ngokukhawuleza ngaphandle kwe-incubation (<300 mg / mL), i-2Rep precipitates (300 mg / mL, 5 mm isikali).c Ixesha le-Gel ye-His-NT2RepCT kunye ne-NT kwi-protein concentrations ebonisiweyo kwi-37 ° C.d Iifoto ze-His-NT2RepCT kunye ne-NT hydrogels kunye nesigcawu kunye nonobumba "NT" oshicilelwe ngaphantsi, ngokulandelelana (zombini 200 mg / mL, isikali bar 5 mm).
IiHydrogels ezenziwe ziiproteni ezahlukeneyo ze-spidroin eziphinda ziphinde ziphinde ziphinde zibe nemibala eyahlukileyo kancinane, kwaye uqwalaselo lweliso lweze lubonisa imigangatho eyahluka-hlukeneyo yokungafihli (Fig. 1b).Iijeli ze-NT zicace kakhulu ngelixa ezinye iijeli zisiba opaque.I-His-NT2RepCT kunye ne-NT iigels eziphonswe kwiityhubhu ze-cylindrical zingasuswa kwi-mold intact (Fig. 1d).
Ukuvavanya ukuba ingaba ijeli yesigcawu sesilika yendalo phantsi kweemeko ngoku ezifunyaniswe zibangela i-gelation yeeproteni ze-spidroin eziphinda-phindaneyo, iingubo zaqokelelwa kwidlala elikhulu le-ampula yesigcawu sebhulorho yaseSweden (Larinioides sclopetarius).Izaleko zagcinwa kwi-20 mM Tris-HCl buffer kwi-50 mg/mL (ngokusekelwe kumlinganiselo wobunzima obomileyo), kodwa akukho gelation yabonwa ngexesha leentsuku ezingama-21 zokufukamela kwi-37 °C (Umfanekiso owongezelelweyo 2a).
Ukulinganisa ezi gels, imilinganiselo ye-rheological ingasetyenziselwa ukufunda inkqubo ye-gelation kunye nokumisela iimpawu zomatshini jikelele.Ngokukodwa, ukubeka iliso kwimodyuli yokugcina (i-elasticity) kwiqondo lokushisa eliphakamileyo linokubonelela ngolwazi malunga nokushisa kwe-gelling kunye neempawu ze-viscoelastic zengubo.Iimvavanyo zokunyuka kobushushu (usebenzisa i-1 ° C / min kwi-25-45 ° C, ngokusekelwe kwizifundo zangaphambili zisebenzisa izisombululo zesitokhwe sesilika yendalo) i-40,41 ibonise ukuba i-moduli yokugcina ye-Hi-NT2RepCT kunye ne-NT solutions yanda ngokunyuka kweqondo lokushisa.yanyuswa (umzobo 2 kunye neFig. 3).Ngokucacileyo, imodyuli ye-NT yaqala ukukhula kwiqondo lokushisa eliphantsi xa kuthelekiswa ne-His-NT2RepCT, ehambelana nexesha elikhawulezayo le-gel elibonwayo xa i-NT ifakwe ngokuthe ngqo kwi-His-NT2RepCT kwi-37 ° C (Umfanekiso 1).Emva kokuhla kweqondo lokushisa okulandelayo, imodyuli yokugcina ayizange ibuyele kumaxabiso aphantsi kwaye yahlala ingaphezulu kwemodyuli yokulahlekelwa (jonga i-Supplementary Fig. 3), ebonisa i-gelation ezinzileyo engenakulinganiswa.Emva kwe-gelation, i-modulus e-elastic yokugqibela isuka kwi-15 ukuya kwi-330 kPa ye-His-NT2RepCT hydrogels kwi-concentration ye-100-500 mg / mL, kunye ne-modulus yokugqibela ye-elastic ye-NT hydrogels (100-500 mg / mL) ukusuka kwi-2 ukuya kwi-1400. kPa (Umfanekiso, 2 kunye nedatha epheleleyo ye-ramp) bona i-Supplementary Fig. 3).
a Utshintsho kwiqondo lokushisa ngexesha lemilinganiselo ye-His-NT2RepCT (300 mg / mL) kunye ne-b NT (300 mg / mL) ngokugungqa.Iintolo zibonisa ukuthambekela kobushushu, kwaye ukukhanya okukhanyayo kwedatha yemodyuli yogcino kubonisa ukuvavanywa kumaxabiso asezantsi etorque yesixhobo kunokuba kuchazwe ngumenzi, nto leyo engunobangela wokwanda kwengxolo.c Ukuphela kwemodyuli yokuqokelela i-His-NT2RepCT kunye ne-NT emva kokushisa okuphakamileyo (100, 300, kunye ne-500 mg / mL).Konke ukufundwa kweemodyuli kuthathwa ngokuphindaphindiweyo kwe-0.1 Hz.
Njengendlela enokwenzeka yokuphanda utshintsho oluhambelana ne-gelation, sirekhode imbonakalo ye-FTIR ye-His-NT2RepCT kunye ne-NT ngaphambi nangemva kwe-gelation kwi-37 ° C (Umfanekiso 3a, b).Njengoko kulindelwe, i-spectra ye-His-NT2RepCT kunye ne-NT izixazululo zihambelana neeprotheni ezibonisa i-α-helix / i-coil engahleliweyo isakhiwo sesibini, kunye nebhendi echazwe kwi-1645 cm-1.Kuzo zombini i-hydrogels, i-gelation ibangele ukubunjwa kweengalo ezimbini phakathi kwe-middle band malunga ne-1617 cm-1 kunye ne-1695 cm-1 (umzobo 3a, b), ebonisa ukubunjwa kwe-antiparallel β-sheet structures.Olu tshintsho lunokubonwa ngokucacileyo kwi-derivative yesibini kunye nokwahlukana kwe-gelation spectra (i-Supplementary Fig. 4b).Iibhanti ezimbini ze-NT β-layer zichazwe ngakumbi kunezo ze-His-NT2RepCT, ebonisa ukuba umxholo opheleleyo we-β-layer bands kwi-NT hydrogel wawuphezulu kune-NT2RepCT hydrogel.
i-spectra yokufunxa ye-FTIR ye-His-NT2RepCT kunye ne-b NT (zombini i-500 mg / mL) ngaphambi (isisombululo) nasemva (ijeli) yokufukamela kwi-37 ° C.c imifanekiso ye-TEM ye-50 mg / ml ye-NT2RepCT ijeli kunye ne-d NT.Isikali bar 200 nm.e Fiber diameters of His-NT2RepCT kunye NT hydrogels.n = 100 imilinganiselo ye-fibrils, p <0.0001.Iibar zempazamo zibonisa ukutenxa okusemgangathweni.Umbindi weempazamo zebar yintsingiselo.Uvavanyo lwe-t olungabhangqwanga (olunemisila emibini) lusetyenziselwe uhlalutyo lwamanani.f I-ThT fluorescence yeeproteni ezahlukeneyo ze-spidroin (100 mg/mL) kuma-37 °C ngaphandle kokungcangcazela.g NT (100 mg/mL) imifuniselo yokugonya evela kwi-100 mg/mL ijeli ye-NT ene-0%, 5%, 10%, kunye ne-20% yembewu.
Uhlalutyo lwe-gel usebenzisa i-electron microscopy yokudluliselwa (TEM) ibonise ukuba i-hydrogel iqukethe i-fibrils efana ne-amyloid (iFig. 3c, 3d).Iifibri ze-NT zenziwe zandiswe (i-5-12 nm ububanzi) kwaye zingenamasebe, ngelixa iifayili ze-His-NT2RepCT zazifutshane ngobude kwaye zibanzi kakhulu ububanzi (7-16 nm) (Umfanekiso 3e).Ezi ziphumo zisivumele ukuba silandele i-kinetics ye-fibrosis usebenzisa i-thioflavin T (ThT) assay.Kuzo zonke iiprotheni ze-spidroin eziphinda ziphinde zakhiwe, isignali ye-fluorescent yanda xa iisampulu zifakwe kwi-37 ° C (Umfanekiso we-3f, i-Fig. 5a eyoNgezelelweyo).Ngokuhambelana noku kufunyaniswayo, ukuhlolwa kwe-microscopic ye-NT kunye ne-His-NT2RepCT phantsi kweemeko ze-gelling kubonise ukunyuka okufanayo kwe-ThT fluorescence ngaphandle kokuqokelelwa kwendawo ebonakalayo ye-ThT-positive aggregates (I-Supplementary Fig. 5b, c).Ukuqulunqwa kwe-fibrils ye-ThT-positive ayizange ihambisane nokunyuka kwe-NT kunye ne-His-NTCT turbidity (i-Supplementary Fig. 5d), oku kuthetha ukuba inethiwekhi ye-fibrils kwi-gel ingenza ngaphandle kokuphazamisa ukucaca kwe-gel.Ukutyalwa ngokudibanisa amanani amancinci eefayili ezenziwe ngaphambili kunokukhawuleza ngokukhawuleza ukubunjwa kwe-fibril kwezinye i-amyloids42,43,44 kodwa ukongeza i-5%, i-10% okanye i-20% (w / w) NT kwisisombululo se-NT hydrocoagulants.isiphumo sembewu (Umfanekiso 3g).Mhlawumbi oku kubangelwa ukuba i-fibrils kwi-hydrogel igxininisekile kwaye ayinakusetyenziswa njengembewu.
Ukuziphatha okungalindelekanga kweeproteni ze-spidroin eziphinda-phindaneyo kumaqondo obushushu aphezulu kuphembelele uphononongo lwembonakalo ye-nuclear magnetic resonance (NMR) ukuchonga utshintsho oluhambelanayo nokwakheka kwejeli.Imbonakalo ye-NMR yezisombululo ze-His-NT2RepCT ezirekhodwe ngokuhamba kwexesha kwi-37 ° C yabonisa ukuba i-CT yayisasongelwe ngokuyinxenye, ngelixa i-NT kunye ne-2Rep izibonakaliso ziye zanyamalala (Umfanekiso we-4a), ebonisa ukuba yayiyi-NT kunye ne-2Rep elawulwa ngokuyinxenye ye-His- NT2RepCT ihydrogel.Isignali ye-CT iphinde yancitshiswa kwi-20% yokuqina kwayo kwasekuqaleni, iphakamisa ukuba i-CT nayo ininzi igxininisekile kwaye ifakwe kwisakhiwo se-hydrogel.Kwinxalenye encinci ye-CT, ehamba njenge-mobile njenge-sample preincubated kwaye ngoko ijongwe yi-NMR yesisombululo, i-spectra ayinayo imiqondiso ye-10 yokuqala yeentsalela ezicwangcisiweyo, mhlawumbi ngenxa yobunzima bokungahambi kakuhle kwenxalenye eqhotyoshelweyo ye-His-NT2Rep.I-spectra ye-NMR ye-imeko ye-hydrogels -NT2RepCT ibonakalise ubukho obuphambili be-α-helices kunye ne-β-layers kwaye, kuncinci, i-coil conformation engahleliwe (Fig. 4b).Uhlalutyo lwe-Chemical shift shift of methionine residues ekhoyo kuphela kwi-NT ibonise ukuba lo mmandla uguqulelwe kwisakhiwo se-β-sheet.Ixesha elixhomekeke kwixesha le-NT kwisisombululo libonise ukuncipha okufanayo kwi-signal intensity (Fig. 4c), kunye ne-NMR eqinile ye-NT hydrogels ibonise ukuba ininzi ye-NT intsalela yaguqulelwa kwizakhiwo ze-β-sheet (Fig. 4d).Ukuhambelana kwe-2Rep akukwazanga ukumiselwa ngokwahlukileyo ngenxa yokuthambekela kwayo ukudibanisa.Nangona kunjalo, i-spectra ye-NMR eqinile ye-NTCT kunye ne-His-NT2RepCT hydrogels ibonakala ifana kakhulu (Umfanekiso we-4b; i-Fig.I-CT hydrogels, i-α-helices, i-β-sheets, kunye nezakhiwo zesibini ze-helical ezingahleliwe zifunyenwe zikhona (i-Supplementary Fig. 6d).Oku kuphakamisa ukuba ezinye iindawo ze-CT zihlala ziyi-α-helices ngelixa ezinye ziba ngama-β-sheets.Ngaloo ndlela, iziphumo ze-NMR spectroscopy zibonisa ukuba i-NT ibalulekile ekubunjweni kwe-hydrogel kwaye iphinde iguqule ibe yi-β-sheet conformation phezu kokudibanisa kunye ne-2Rep kunye ne-CT.Ngokuhambelana nale nto, kutshanje sifumene ukuba i-zippers yendawo ye-amyloid inokwenzeka ukuba yenze kuzo zonke ii-helices ezintlanu ze-NT domain, kwaye i-algorithm ye-Waltz iqikelele ummandla we-amyloidogenic kwi-helix 1 (umzobo 4e).
I-2D spectra ye-15N-HSQC 10 mg / mL Isisombululo se-His-NT2RepCT ngaphambi (eluhlaza okwesibhakabhaka) kunye neeyure ze-19 emva kokufukamela (obomvu) kwi-37 ° C.Iincopho zomnqamlezo ngamnye kwi-spectrum ebomvu kunye ne-F24, i-G136, i-polyA kwi-spectrum eluhlaza okwesibhakabhaka ibonakaliswe ngonobumba omnye weempawu ze-amino acid kunye namanani aseleyo.I-insets ibonisa ukuxhomekeka kokuqina komqondiso ngexesha leentsalela ezikhethiweyo ezivela kwi-NT, 2Rep, kunye ne-CT domains.b I-Solid-state radiofrequency (RFDR) imbonakalo ye-His-NT2RepCT hydrogels.Ulungelelwaniso lweentsalela ze-Cα / Cβ ezibonwe kwi-spectra ye-RFDR zigqitywe ngokuthelekiswa nomzekelo we-peptide utshintsho lweekhemikhali kunye namaxabiso athathwe kwi-statistics82,83 kunye nezakhiwo zabo zesibini.I-SSB – ibhanti elisecaleni elijikelezayo.c I-One-dimensional spectra ye-15N-HSQC 10 mg/mL isisombululo se-NT ngexesha lokufukamela kwi-37 °C kwiiyure ezingama-36.I-inset ibonisa ukuqina kwevolumetric xa kuthelekiswa nexesha.d Imeko eqinileyo yeRFDR spectra ye-NT hydrogels.Ukulungelelaniswa kweentsalela ze-Cα / Cβ kunye nezakhiwo zabo zesibini eziqatshelwe kwi-spectra ye-RFDR zibonisiwe.e Ngokusekelwe kwiprofayili ye-NT45.79 ye-fibrillation propensity evela kwi-database ye-Zipper (https://services.mbi.ucla.edu/zipperdb/).Amandla eRosetta yefestile yokutshintsha kombane we-hexapeptide iboniswe kwi-kcal/mol.Imivalo ebomvu ibonisa i-hexapeptides ene-fibrosis propensity ephezulu (amandla e-Rosetta angaphantsi kwe-23 kcal / mol; ngaphantsi komgca onamachaphaza).Imivalo eluhlaza ibonisa amaqhekeza anamandla eRosetta ngaphezu komgubasi ngoko ke akufane kwenzeke ukuba zenze iziphu eziqinileyo.Iziqwenga eziqukethe iproline zazingabandakanywa kuhlalutyo (ngaphandle kwamakholomu).Izikwere zibonisa iindawo ze-amyloidosis ezixelwe kwangaphambili yi-Waltz algorithm81 (https://waltz.switchlab.org).Ukulandelelana kweentsalela ze-amino acid ze-NT ziphezulu, kwaye iintlobo zeentsalela ezifunyenwe kwisakhiwo sesibini se-β (esinqunywe yi-slid-state NMR spectroscopy) iboniswe ngobomvu.Izikhundla ze-NT α-helices ezintlanu zichongwe njenge (H1-H5)28.
Kwi-pH <6.5, i-HT iyancipha, ikwazi ukumelana nobushushu-okanye i-urea-induced denaturation18.Ukucacisa indlela i-NT dimerization kunye nokuzinza kuchaphazela ngayo i-gelation, izisombululo eziqukethe i-100 mg / ml NT zilawulwa kwi-pH 8, 7, kunye ne-6 usebenzisa uvavanyo lwe-vial inversion.Iisampuli ze-NT zifakwe kwi-pH 8 kunye ne-7 ye-gelled emva kwe-30 min kwi-37 ° C, kodwa i-pH 8 ijeli yahlala icacile, ngelixa i-pH 7 ijeli ibonisa i-precipitate ebonakalayo (Umfanekiso 5a).Ngokwahlukileyo, isisombululo esine-HT kwi-pH 6 asizange senze i-gel, kwaye i-precipitate enkulu ingabonwa emva kwe-20 min kwi-37 ° C.Oku kuphakamisa ukuba i-dimers ngokwabo kunye / okanye ukuzinza kwabo okuphezulu xa kuthelekiswa ne-monomers kuthintela i-gelation.Ukuqulunqwa kwemvula ye-NT kwi-pH 7 kunye ne-6 bekungalindelekanga, kuba kuye kwaxelwa ukuba i-NT inyibilika kwi-200 mg / ml27, iphinda iphinde ibuyele emva kokutshatyalaliswa kobushushu, kwaye igcina i-α-helix kumaxabiso aphantsi pH 18. Inkcazo enokwenzeka yezi zintlukwano kukuba iimvavanyo ezichazwe ngaphambili zenziwa kwiqondo lokushisa elisezantsi okanye ngaphantsi, okanye kwiiprotheyini eziphantsi kakhulu16,18,19.
Uvavanyo lwe-NT ye-vial inversion (100 mg/mL) kwi-pH 8, 7, 6 kunye ne-154 mM NaCl (pH 8) emva kokufukamela kwi-37°C.b I-CD spectra ye-NT ene-154 mM NaF kunye ne-154 mM NaCl, ngokulandelelanayo.I-Molar ellipticity kwi-222 nm iguqulwa ibe ngumlinganiselo we-folds yendalo.c NT inversion assay (100 mg/mL) NT * (37 °C kunye 60 °C), NTA72R (37 °C), kunye His-NT-L6 (37 °C kunye 60 °C).d i-CD spectra ye-NT mutants NT*, NTA72R, kunye ne-His-NT-L6.I-Molar ellipticity kwi-222 nm iguqulwa ibe ngumlinganiselo we-folds yendalo.e Uvavanyo lokuguqula i-NTFlSp, i-NTMiSp kunye nokunciphisa i-NTMiSp (100 mg / mL).Isikali bar 5 mm.f I-CD spectra ye-NT, NTFlSp, NTMiSp kunye ne-NTMiSp encitshisiweyo.I-Molar ellipticity kwi-222 nm iguqulwa ibe ngumlinganiselo we-folds yendalo.Umboniso ogcweleyo we-NT kuma-25 °C kunye nama-95 °C uboniswa kuMfanekiso oSongezelelo wesi-8.
Ukugxininiswa kwetyuwa ye-physiological imisela intsebenziswano ye-electrostatic phakathi kwee-subunits ze-NT kunye ne-dimerization yokudluliselwa kwe-NT ukuya kwi-pH18 ephantsi.Sifumene ukuba ubukho be-154 mM NaCl kunye ne-NaF ngokwenene inhibit i-gelation, ngokulandelanayo (Umfanekiso 5a, b; Umzobo owongezelelweyo we-2b) kwaye ezi tyuwa zandisa ukuzinza kwe-thermal ye-NT monomers (umzobo 5b, i-Fig. 8). .Kwakhona kuphakamisa ukuba ukuphuculwa kozinzo, kunokuba i-dimerization, ithintele ukubunjwa kwe-gel.
Ukuphonononga ngakumbi indima ye-protein dimerization kunye nokuzinza kwi-gelation, sasebenzisa ii-mutants ezimbini, i-NT * kunye ne-NTA72R, ehlala i-monomeric kwi-pH28.30 ephantsi.I-NT* yintlawulo ephindwe kabini yokuguqula i-mutant apho ukuhanjiswa kwentlawulo ebonakalayo ye-dipole ye-monomer ithotywe, ethintela u-dimerization kunye nokwandisa kakhulu ukuzinza kwe-monomer.I-NTA72R yi-dipole ehlawuliswayo, kodwa i-Arg-ethathelwe endaweni ye-Ala ikumda we-dimer, ngoko ke iinguqulelo zenguquko ziphazamisa unxibelelwano lwe-subunit olufunekayo ukuze kunciphe.Emva kokufakwa kwi-37 ° C, i-NT * ayizange yenze i-hydrogel, ngelixa i-NTA72R yenza i-gel opaque ye-15 min (umzobo 5c).Ekubeni zombini i-NT * kunye ne-NTA72R ayikwazi ukucima kodwa ihluke kwi-monomer stability (Fig. 5d), ezi ziphumo zibonisa ngokucacileyo ukuba ukuzinza okuphezulu kwe-thermodynamic kuthintela i-NT kwi-gelling.Oku kuxhaswa kwakhona kukuba i-HT * yenza i-gel xa ingazinzile kwiqondo lokushisa eliphezulu (emva kwe-8 min kwi-60 ° C; Umzobo 5c).Ngaphambili kuye kwaboniswa ukuba umxholo ophezulu we-methionine kwi-NT unyibilikisa ukusonga kwayo kwendalo kunye nokuba i-Met emithandathu kwi-Leu substitutes (ekubhekiselwe kuyo apha njenge-His-NT-L6) iqinisa ngamandla i-monoma ye-NT46.Ngokusekelwe kwingcinga yokuba ukuguquguquka kwesakhiwo kuyadingeka kwi-NT gel ukubunjwa, sifumene ukuba i-His-NT-L6 i-stable mutant ayizange ifakwe kwi-37 ° C (Umfanekiso 5c, d).Nangona kunjalo, i-His-NT-L6 iphinde yenza i-gel kwi-incubation kwi-60 ° С kwimizuzu engama-60 (umzobo 5c).
Ukukwazi kwe-NT ukuguqula kwizakhiwo ze-β-sheet kunye nokwenza ii-hydrogels kubonakala kusebenza kwezinye kodwa kungekhona zonke iindawo ze-NT ze-spidroin.Ii-NTs ezisuka kwiintlobo ezahlukeneyo zesilika kunye neentlobo zezigcawu, i-Trichonephila clavipes (NTFlSp), zenze iijeli nangona zinemethionine ephantsi kunye nozinzo oluphezulu lwe-thermal (Umfanekiso 5e, f kunye neTheyibhile eyoNgezelelweyo yesi-2).Ngokwahlukileyo, i-NT evela kwi-ampullar protein spidroin encinci esuka kwi-Araneus ventricosus (NTMiSp) ene-thermal stability kunye nomxholo ophezulu we-methionine ayizange yenze i-hydrogels (i-Supplementary Table 2 kunye ne-Fig. 5e, f).Le yokugqibela inokudityaniswa nobukho be-intramolecular disulfide bonds29,47.Ngokuqhubekayo, xa iibhondi ze-disulfide ze-NTMiSp zancitshiswa, zakha i-hydrogel emva kokufakwa kwi-37 ° C kwi-10 min (umzobo 5e).Ukuqukumbela, kufuneka kuqatshelwe ukuba ukuguquguquka kwesakhiwo kubalulekile, kodwa kungekhona kuphela, umgaqo wokuqulunqwa kwejel evela kwi-NT.Enye into enokubaluleka kukuthambekela ukwenza iifibri ze-amyloid, kunye nohlalutyo kunye nedatha ye-zipper kunye ne-algorithm ye-Waltz ibonise ulungelelwaniso phakathi kokukwazi ukwenza iigels kunye nobukho bemimandla ye-amyloidogenic, kunye nobungakanani bemimandla eqikelelweyo. ukwenza iziphu zesteric.Kwakukho ulungelelwaniso (iThebhile eyoNgezelelweyo 2 kunye neFig. 9 eyoNgezelelweyo).
Ukukwazi kwe-NT ukwenza ii-fibrils kunye nokwenza iijeli phantsi kweemeko ezithandekayo kwasikhokelela ekubeni sicinge ukuba ukudibanisa kwe-NT kunye namanye amaqhekeza eprotheyini kusenokwenza iijeli ezinomsebenzi opheleleyo wamaqabane okudibanisa.Ukuvavanya oku, sazisa iprotheni ye-fluorescent eluhlaza (GFP) kunye ne-purine nucleoside phosphorylase (PNP) kwi-C-terminus ye-NT, ngokulandelanayo.Iiprotheni ezibangelwa yi-fusion zibonakaliswe kwi-E. coli kunye nezivuno eziphezulu kakhulu zokugqibela (i-150 mg / L kunye ne-256 mg / L iinkcubeko ze-flask ze-His-NT-GFP kunye ne-His-NT-PNP, ngokulandelanayo), ngokuhambelana noko kubonisiwe. Yezinye iiproteni ezidityaniswe kwi-NT Ref.30. I-His-NT-GFP (300mg / mL) kunye ne-His-NT-PNP (100mg / mL) iiprotheyini ze-fusion zenza ii-gel emva kweeyure ze-2 kunye neeyure ze-6.5 kwi-37 ° C kwaye, ngokubalulekileyo, i-fraction ye-GFP yahlala ingatshintshi.ijongwe emva kokuxutywa, nge>70% yokuqina kwe-fluorescence yokuqala eseleyo emva kokuxutywa (Umfanekiso 6a).Ukulinganisa umsebenzi we-PNP kwiisombululo zakhe ze-NT-PNP kunye neegels, kwafuneka sihlambulule iprotheni ye-fusion kunye ne-NT ngenxa yokuba umsebenzi we-enzymatic wokulungiswa okucocekileyo wawungaphandle koluhlu lokufumanisa i-assay kwi-concentrations ye-gelling.I-gel eyenziwe ngomxube oqulethe i-0.01 mg / mL I-His-NT-PNP kunye ne-100 mg / mL NT igcine i-65% yomsebenzi wokuqala we-enzymatic weesampuli zangaphambili (umzobo 6b).I-gel yahlala ilungile ngexesha lokulinganisa (i-Supplementary Fig. 10).
i-Relative fluorescence intensity ngaphambi nangemva kwe-gelation ye-His-NT-GFP (300 mg / mL) kunye ne-vial eguquliweyo equlethe i-Hidrogel ye-His-NT-GFP (300 mg / mL) phantsi kokukhanya okubonakalayo kunye ne-UV.Amanqaku abonisa imilinganiselo yomntu ngamnye (n = 3), imivalo yempazamo ibonisa ukutenxa okuqhelekileyo.Ixabiso eliphakathi liboniswe embindini weempazamo zebar.b Umsebenzi we-PNP ufunyenwe ngohlalutyo lwe-fluorometric usebenzisa izisombululo kunye neegels ezibandakanya i-NT (100 mg / ml) kunye nomxube oqukethe i-0.01 mg / ml yakhe-NT-PNP kunye ne-100 mg / ml iidola ezintsha zaseTaiwan.I-inset ibonisa i-vial eguqulweyo equlethe i-hydrogel equlethe i-His-NT-PNP (i-5 mm isikali bar).
Apha, sixela ukubunjwa kwe-hydrogels ukusuka kwi-NT kunye nezinye iiprotheni ze-spidroin eziphinda ziphinde zifakwe ngokufaka isisombululo seprotheni kwi-37 ° C (Umfanekiso 1).Sibonisa ukuba i-gelation ihambelana nokuguqulwa kwe-α-helices kwi-β-layers kunye nokwakhiwa kwee-fibrils ezifana ne-amyloid (Imifanekiso 3 kunye ne-4).Oku kufunyaniswayo kuyamangalisa njengoko i-NTs i-coiled globular five-helix bundles eyaziwa ngokuba yi-solubility ephezulu kakhulu kunye nokuzinza okuphezulu kwi-concentrations> 200 mg / mL kwi-4 ° C iintsuku eziliqela27.Ukongeza, i-NTs iphinda iphindeke emva kokutshintsha kobushushu kwindawo ephantsi yeprotheyini kwi-µM.Ngokweziphumo zethu, ukubunjwa kwe-fibril kufuna ukudibanisa> i-10 mg / mL iprotheni yoxinzelelo kunye nokushisa okuphakamileyo kancinci (Umfanekiso 1).Oku kuhambelana nengcamango yokuba i-amyloid fibrils inokuthi yenze kwiiprotheni ezigoqiweyo ze-globular ezikwimeko engabonakaliyo ngenxa yokutshintsha kwe-thermal phantsi kweemeko ze-physiological 48.Imizekelo yeeprotheni ezifumana olu kuguqulwa ziquka i-insulin49,50, β2-microglobulin, i-transthyretin kunye ne-lysozyme51,52,53.Nangona i-NT i-α-helix kwilizwe layo lokuzalwa, malunga ne-65% ye-polypeptide chain iyahambelana nokwakheka kwe-zipper ye-steric (Fig. 4e) 45.Ekubeni i-monomer ihamba ngokuguquguqukayo i-mobile46, inokutyhila le mimandla ye-amyloidogenic enokubakho kumaqondo obushushu aphakamileyo kunye noxinzelelo oluphezulu lweprotheyini epheleleyo inokufikelela kugxininiso olubalulekileyo lwe-amyloid fibril formation54.Ukulandela le ngqiqo, sifumene ulungelelwaniso olungalunganga phakathi koxinzelelo lwe-spidroin kunye nexesha le-gelation (umzobo 1c), kwaye ukuba i-monomeric NT conformation iyazinziswa mhlawumbi ngokuguquguquka (NT *, His-NT-L6) okanye ngokongezwa kwetyuwa, kunokuthintela ukubunjwa kwe-hydrogels (umzobo 5).
Kwiimeko ezininzi, i-amyloid fibrils iyanyamalala kwisisombululo njenge-precipitate, kodwa phantsi kweemeko ezithile zingenza i-hydrogels55,56,57.I-Hydrogel-forming fibrils idla ngokuba ne-aspect ratio ephezulu kwaye yenza i-stable-dimensional network networks ngokusebenzisa i-molecular entanglement, i-55,58 ihambelana neziphumo zethu.Ukwenziwa kwe-hydrogel kwi-vitro, iiprotheyini zihlala zihlanjululwa ngokupheleleyo okanye zincinci, umzekelo, ngokuvezwa kwi-solvents eziphilayo, ukushisa okuphezulu (70-90 ° C) kunye / okanye i-pH ephantsi (1.5-3.0) 59,60,61,62.I-spidroin hydrogels echazwe apha ayifuni ukucutshungulwa rhabaxa, kwaye ayifuni i-agent edibanisa ukunqumlana ukuzinzisa ii-hydrogels.
Ngaphambili kuye kwaxelwa ukuba i-spidroin iphinda kunye nee-QDs, ezibonakala zingena kwi-β-sheet switching ngexesha le-silk spinning, yenza i-hydrogels.Xa kuthelekiswa neziphumo zethu, amaxesha okufukamela kunye/okanye amaqondo obushushu okufukamela ayemade kakhulu okanye aphezulu, ngokulandelelanayo, kwaye iziphumo ze-hydrogels bezisoloko zi-opaque (Umfanekiso 7 kunye neTheyibhile eyoNgezelelweyo 1) 37, 38, 63, 64, 65, 66, 67, 68 , 69. Ukongeza kumaxesha ejeli akhawulezayo, i-NT hydrogels>300 mg/mL (30%) igqwesile zonke ezinye ezichazwe kwakhona ezihlaziyiweyo ze-spider silk protein hydrogels, kunye nee-hydrogel zendalo ezifana ne-gelatin, i-alginate (2%), i-agar (0.5 % ) kunye ne-collagen.(0.6%) (Umfanekiso 7 kunye neTheyibhile ezongezelelweyo 1 kunye ne-3) 37,39,66,67,68,69,70,71,72,73,74.
Ixesha lejeli kunye ne-elastic modulus ye-hydrogels kolu phononongo yathelekiswa nezinye ii-hydrogels ezisekelwe kwi-spidroin kunye nee-hydrogel zendalo ezikhethiweyo.Iireferensi zinikwe kunye nenkcazo yeemeko ze-gelation.APS Ammonium persulfate, ubushushu begumbi.Idatha 37, 38, 39, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
Kubonakala ngathi izigcawu ziye zavelisa iindlela zokuthintela ukuba i-spidroin ingageli ngexesha lokugcinwa kwayo.Nangona i-protein ephezulu yeprotheyini kwi-silk gland, ummandla omkhulu wokuphindaphinda ohambelana ne-domain ye-terminal uthetha ukuba ukuxinwa okubonakalayo kwe-NT kunye ne-CT kwi-gland ihambelana malunga ne-10-20 mg / ml, kumda wolu cwaningo.efunekayo kwi-in vitro yaqaphela ukwakheka kwe-hydrogel.Ukongezelela, ukugxininiswa okufanayo kweetyuwa 16 kuzinzile i-NT, njengamadlala esilika (umzobo 5b).Ukuhambelana kwe-NT kuye kwaphononongwa kwi-E. coli cytosol kwaye kwafunyaniswa ukuba isongelwe ngokuqinileyo kunaxa ivavanywa kwi-vitro, ebonisa ngakumbi ukuba ityuwa okanye ezinye izinto zithintela ukudityaniswa kwayo kwi-vivo.Nangona kunjalo, ukukwazi kwe-NTs ukuguqula kwi-β-sheet fibrils kunokubaluleka ekwenzeni i-filament kwaye kufuneka kuphandwe kwizifundo ezizayo.
Ukongeza kwimiba entsha ye-NT-amyloid-efana ne-fibril kunye ne-hydrogel formation ebonwe kolu cwaningo, sibonisa kwakhona ukuba le nto ingaba nezicelo ze-biotechnological kunye ne-biomedical (Fig. 8).Njengobungqina bengcamango, sidibanise i-NT kunye ne-GFP okanye i-PNP kwaye sabonisa ukuba iprotheni ye-fusion iphinda ifake i-hydrogels xa ifakwe kwi-37 ° C kunye nokuba i-GFP kunye ne-PNP iiqhezu zigcina kakhulu umsebenzi wazo emva kwe-gelation (Umfanekiso 6).I-Nucleoside phosphorylases zibalulekile i-catalysts synthesis ye-nucleoside analogues75, eyenza ukufumanisa kwethu kuhambelana neshishini le-biopharmaceutical.Ingqikelelo yokubonakalisa iiprotheyini ezidityanisiweyo ezenza ii-hydrogels ezicacileyo phantsi kweemeko ezifanelekileyo zivumela ukudalwa kwee-hydrogels ezisebenzayo ezineempawu ezithandekayo kuluhlu olubanzi lwezicelo ezinje nge-enzyme immobilization, ukukhutshwa kweziyobisi okulawulwayo kunye nobunjineli bezicubu.Ukongeza, i-NT kunye ne-NT* ziziphawuli ezisebenzayo zokubonisa30, okuthetha ukuba i-NT kunye neentlobo zayo zingasetyenziselwa ukuveliswa okuphezulu kweeprotheni ezidibeneyo ezinyibilikayo kunye nokudalwa okulandelayo kweeprotheyini ezijoliswe kuzo ezingashukumiyo kwi-3D hydrogels.
I-NT iyanyibilika, i-α-helical kwaye izinzile kwiindawo eziphantsi (µM) kunye ne-37 ° C.Kwiqondo lokushisa elifanayo, kodwa ekunyuseni okunyukayo (> 10 mg / ml), i-NT yenza iigels ezibandakanya i-amyloid-like fibrils.Iiprotheyini ze-NT fusion nazo zenza iigels ze-fibrillar ezinamaqhekeza adibeneyo asebenzayo, avumela iiprotheni ezahlukeneyo ukuba zingashukunyiswa kwi-3D hydrogels usebenzisa i-NT.Ngezantsi: i-NT (PDB: 4FBS) kunye nemifanekiso ye-fiber networks kunye nezakhiwo zeprotheyini ezinxulumene nazo (zicingelwa kwaye zingatsalwanga kwisikali, i-GFP PDB: 2B3Q, 10.2210 / pdb2B3Q / pdb; PNP PDB: 4RJ2, 10.2210 / pdb4bJ2).
Ulwakhiwo (jonga i-Supplementary Table 4 yoluhlu olupheleleyo olubandakanya ulandelelwano lwe-amino acid) luye lwahlanganiswa kwi-plasmid pT7 kwaye yaguqulwa yaba yi-E. coli BL21 (DE3).I-E. coli equkethe i-plasmids yobunjineli yayifakwe kwi-Luria broth yongezwa nge-kanamycin (70 mg / l) kwaye ikhule ngobusuku kwi-30 ° C kunye ne-250 rpm.Emva koko inkcubeko ifakwe i-1 / 100 kwi-LB medium equkethe i-kanamycin kwaye ikhuliswe kwi-30 ° C kunye ne-110 rpm de i-OD600 ifike kwi-0.8.Kwizifundo ze-NMR, iibhaktheriya zakhuliswa kwi-M9 encinci ye-medium equkethe i-2 g ye-D-glucose 13C (Aldrich) kunye ne-1 g ye-ammonium chloride 15N (i-Cambridge Isotope Laboratories, Inc.) yokubhala iprotheni ene-isotopi.Yehlisa iqondo lobushushu ukuya kuma 20 degrees celcius kwaye wenze ukubonakaliswa kweprotheyini nge-0.15 mM isopropylthiogalactopyranoside (uxinzelelo lokugqibela).Emva kokubonakaliswa kweprotheni ebusuku, iiseli zavunwa kwi-7278 × g, 4 ° C kwi-20 min.Iipilisi zeeseli zaphinda zamiswa kwi-20 mM Tris-HCl, pH 8, kwaye zakhenkcezwa de zisetyenziswe ngakumbi.Iiseli ezincibilikisiweyo zihlanjululwe kusetyenziswa i-cell disruptor (i-TS series machines, Constant Systems Limited, eNgilani) kwi-30 kPa.Emva koko iilysates zafakwa kwi-centrifuged kwi-25,000 g imizuzu engama-30 kwi-4 ° C.Kwi-NTMiSp, i-pellet yaphinda yamiswa kwi-2 M urea, i-20 mM Tris-HCl, i-pH 8, kunye ne-sonicated ye-2 min (2 s on / off, 65%), emva koko i-centrifuged kwakhona kwi-25,000 xg, 4 ° C. ngaphakathi 30 imiz.I-supernatant yalayishwa kwikholamu ye-Ni-NTA, ihlanjwe nge-20 mM Tris-HCl, i-2 mM imidazole, i-pH 8, kwaye ekugqibeleni iprotheni yahluthwa nge-20 mM Tris-HCl, 200 mM imidazole, pH 8. Ukuvelisa i-NT2RepCT kunye I-NTCT, i-thrombin digestion yazisa indawo (ThrCleav) phakathi kweYakhe kunye ne-NT.Iindawo ze-Thrombin cleavage zikhona kwakhona kwi-His-NT-ThrCleav-2Rep (ivelisa i-2Rep), i-His-thioredoxin-ThrCleav-NT (ivelisa i-NT), i-His-thioredoxin-ThrCleav-CT (ivelisa i-CT), i-His-Thioredoxin-ThrCleav-NT .* (ivelisa i-NT *), i-His-Thioredoxin-ThrCleav-NTA72R (ivelisa i-NTA72R), i-His-Thioredoxin-ThrCleav-NTFlSp (ivelisa i-NTF1Sp), kunye ne-His-Sulphur Redoxin-ThrCleav-NTMiSp (ivelisa i-NTMiSp).Ukwakhiwa kwaxutywa nge-thrombin (1: 1000) kwaye i-dialyzed ngobusuku kwi-4 ° C. kunye ne-20 mM Tris-HCl, pH 8, usebenzisa i-Spectra / Por dialysis membrane ene-molecular weight threshold ye-6-8 kDa.Emva kwe-dialysis, isisombululo silayishwa kwikholomu ye-Ni-NTA kwaye i-effluent equkethe iprotheni enomdla iqokelelwa.Ugxininiso lweprotheyini luye lwamiselwa ngokulinganisa i-UV yokufunxa kwi-280 nm usebenzisa i-coefficient yokuphela kweprotheni nganye, ngaphandle kwe-NTF1Sp, esebenzisa i-Bradford assay ngokweprotocol yomenzi.Ubunyulu bugqitywe yi-SDS polyacrylamide (4–20%) i-gel electrophoresis kunye ne-Coomassie brilliant blue staining.Iiprotheyini zigxininiswe ngokusebenzisa izihlungi ze-centrifuge (VivaSpin 20, GE Healthcare) kwi-4000 xg kunye ne-10 kDa i-molecular weight cutoff kwi-20 imijikelezo yemizuzu.
Kunyibilika isisombululo seproteni kunye nombhobho ngononophelo i-150 µl kwi-1 ml ecacileyo ye-septum vial (8 x 40 mm Thermo Scientific).Iityhubhu zavalwa kwaye zavalwa ngeparafilm ukuthintela ukuvela kwamanzi.Iisampulu (n = 3) zifakwe kwi-37 ° C okanye i-60 ° C kwaye ziguqulwe ngezikhathi ezithile ukuze ziqwalasele i-gelation.Iisampulu ezingazange zifakwe ijeli zifukanywe iveki enye ubuncinane.Nciphisa iibhondi ze-disulfide ze-NTMiSp nge-10 mM DTT nge-10 µM yeprotheni.Ukuhlalutya i-gelation yesigcawu sesilika yendalo, isigcawu sebhulorho yaseSweden yasikwa, amadlala amabini akhululweyo abekwe kwi-200 μl ye-20 mM Tris-HCl buffer pH 8 kwaye yasikwa ukuvumela ukugquma ukuba kwahlukane namadlala..Imixholo yamadlala inyibilika kwi-buffer, i-50 µl yokumisela ubunzima obomileyo (ngokufukanyelwa kweengqayi ezivulekileyo kuma-60 °C ukuya kubunzima obungaguqukiyo) kunye ne-150 µl yegelation kuma-37 °C.
Ijometri yokulinganisa / isixhobo senziwe ngensimbi engenasici kusetyenziswa ipleyiti ehambelanayo kunye nobubanzi obuphezulu be-20 mm kunye ne-gap ye-0.5 mm.Fudumeza isampulu ukusuka kwi-25 °C ukuya ku-45 °C kwaye ubuyele kwi-25 °C ngesantya se-1 °C ngomzuzu usebenzisa ipleyiti yentsimbi engenastainless ezantsi kwePeltier.Imilinganiselo ye-Vibrational iqhutywe kwi-frequency ye-0.1 Hz kunye nommandla we-viscoelastic wezinto eziphathekayo kwi-strain ye-5% kunye ne-0.5% yeesampuli ze-100 mg / mL kunye ne-300-500 mg / mL, ngokulandelanayo.Sebenzisa igumbi lokufuma eliqhelekileyo ukunqanda ukuguquka kwamanzi.Idatha yahlalutywa kusetyenziswa iPrism 9.
Ukuqokelela i-infrared (IR) i-spectra kwiqondo lokushisa ukusuka kwi-800 ukuya kwi-3900 cm-1.Isixhobo se-ATR, kunye nendlela yokukhanya kwi-spectrometer, ihlanjululwa ngomoya owomileyo ococekileyo ngaphambi nangexesha lovavanyo.Izisombululo (i-500 mg / mL ukunciphisa iindawo eziphakamileyo zokufunxa amanzi kwi-spectra) zifakwe kwimibhobho kwiikristale, kwaye iigels (500 mg / mL) zenziwe ngaphambi kokulinganisa kwaye emva koko zidluliselwe kwiikristale (n = 3).Izikena ze-1000 zirekhodwe ngesisombululo se-2 cm-1 kunye nomjikelezo we-zero we-2. I-derivative yesibini ibalwa ngokusebenzisa i-OPUS (i-Bruker) isebenzisa uluhlu olululayo lwamanqaku alithoba.I-spectra yayiqhelekileyo kwindawo efanayo yokudibanisa phakathi kwe-1720 kunye ne-1580 cm-1 usebenzisa i-F. Menges "Spectragryph - Optical Spectroscopy Software".Kwi-ATR-IR spectroscopy, ubunzulu bokungena bomqadi we-infrared kwisampulu kuxhomekeke kwinani lamaza, okukhokelela ekufunxeni okunamandla kumanani amaza asezantsi kunamanani aphezulu amaza.Ezi ziphumo azizange zilungiswe kwi-spectra eboniswe kwiMifanekiso.3 kuba zincinci kakhulu (Fig. 4).I-spectra echanekileyo yalo mzobo ibalwe kusetyenziswa i-software ye-Bruker OPUS.
Ngokomgaqo, i-quantification ebanzi yokuguqulwa kweeprotheyini inokwenzeka emva kokuchithwa okuthembekileyo kwamacandelo ngaphakathi kwe-amide I peak.Nangona kunjalo, eminye imiqobo ivela ekusebenzeni.Ingxolo kwi-spectrum inokuvela njengeencopho (ezingeyonyani) ngexesha le-deconvolution.Ukongeza, incopho ngenxa yokugoba kwamanzi ingqamana nendawo ye-amide I peak kwaye inokuba nobukhulu obufanayo beesampulu eziqulethe isixa esikhulu samanzi, njengejeli enamanzi efundwa apha.Ngoko ke, asizange sizame ukubola ngokupheleleyo i-amide I peak, kwaye ukujonga kwethu kufuneka kuqwalaselwe kuphela ukuxhasa ezinye iindlela ezifana ne-NMR spectroscopy.
Izisombululo ze-50 mg / ml NT kunye ne-His-NT2RepCT zifakwe ebusuku kwi-37 ° C.Emva koko i-hydrogel yahlanjululwa nge-20 mM Tris-HCl (pH 8) kwi-concentration ye-12.5 mg / ml, ixutywe kakuhle kwaye ifakwe umbhobho ukuze iphule i-gel.Emva koko, i-hydrogel yahlanjululwa ngamaxesha angama-10 kunye ne-20 mM Tris-HCl (pH 8), i-5 μl yesampuli isetyenziswe kwigridi yobhedu ehlanganiswe ne-formvar, kwaye isampuli engaphezulu isuswe ngephepha lokuqhawula.Iisampulu zihlanjwe kabini nge-5 µl ye-MilliQ yamanzi kwaye ihlanjwe nge-1% ye-uranyl formate imizuzu emi-5.Susa ibala eligqithisileyo ngephepha elifunxayo, uze womise umoya umnatha.Imifanekiso yenziwa kwezi grids kusetyenziswa iFEI Tecnai 12 Spirit BioTWIN esebenza nge-100 kV.Imifanekiso irekhodwe kwi-x 26,500 kunye ne-x 43,000 yokukhulisa usebenzisa ikhamera ye-CCD ye-Veleta 2k × 2k (i-Olympus Soft Imaging Solutions, GmbH, Münster, eJamani).Kwisampula nganye (n = 1), imifanekiso ye-10-15 irekhodwe.I-ImageJ (https://imagej.nih.gov/) yayisetyenziselwa uhlalutyo lomfanekiso kunye nokulinganisa i-fiber diameters (n = 100, iifayili ezahlukeneyo).I-Prism 9 yayisetyenziselwa ukwenza iimvavanyo ze-t ezingenakulinganiswa (ezinemisila emibini).Intsingiselo ye-His-NT2RepCT kunye ne-NT fibrils yayiyi-11.43 (SD 2.035) kunye ne-7.67 (SD 1.389) nm, ngokulandelanayo.Ixesha lokuzithemba (95%) ngu-4.246 ukuya ku-3.275.izidanga zenkululeko = 198, p <0.0001.
I-80 µl yeesampulu zolwelo eziqulathe i-10 µM thioflavin T (ThT) zilinganiswe ngee-triplicate (n = 3) phantsi kweemeko ezimileyo kusetyenziswa i-Corning 96-icwecwe elisezantsi elimnyama elisezantsi elicacileyo (i-Corning Glass 3881, e-USA).Iintlukwano ze-Fluorescence zirekhodwe kusetyenziswa i-440 nm yokucoca i-excitation filter kunye ne-480 nm emission filter (i-FLUOStar Galaxy esuka kwi-BMG Labtech, e-Offenburg, eJamani).Umqondiso we-ThT awuzange ugcwaliswe okanye ucinywe, njengoko iimvavanyo ezinogxininiso olwahlukeneyo lwe-ThT lwenziwa ngaphandle kokutshintsha ubungakanani bomqondiso.Rekhoda ukufunxa kwi-360 nm kumlinganiselo wenkungu.Kwimifuniselo yembewu, i-100 mg/mL iijeli zenziwa kwi-37° C., zaphinda zamiswa, zaze zasetyenziselwa ukuhlwayelwa kwembewu kwi-molar ratios ye-5%, 10%, kunye ne-20%.Idatha yahlalutywa kusetyenziswa iPrism 9.
Thaw stocks of His-NT2RepCT and NT >100 mg/mL kumkhenkce kwaye uhluze ngesihluzo se-0.22 µm.Ugxininiso lubalwe ngokulinganisa i-absorbence kwi-280 nm usebenzisa i-Nanodrop.Kwimithombo ye-96-well black plate non-binding (Corning) kunye nezantsi ecacileyo, iisampulu zihlanjululwe kwi-20 mg / ml kwi-20 mM Tris-HCl pH 8 kwaye ixutywe ne-5 μM ThT (ingqwalasela yokugqibela), isampuli epheleleyo 50 μl umthamo.Iisampulu zafanekiswa yonke imizuzu ye-10 kwi-37 ° C kwi-microscope ye-CellObserver (Zeiss) enejelo lokukhanya eligqithisiweyo kunye ne-FITC yokuvuselela kunye neeseti zokucoca ukukhutshwa kwe-ThT imaging.I-20x / 0.4 lens isetyenziselwa ukwenza umfanekiso.IZen Blue (Zeiss) kunye ne-ImageJ (https://imagej.nih.gov/) zisetyenziselwe uhlalutyo lwemifanekiso.Iigel nazo zalungiswa ukusuka kwi-NT kunye ne-His-NT2RepCT isisombululo kwi-concentration ye-50 mg / mL equkethe i-20 mM Tris pH 8 kunye ne-5 µM ThT kwaye ifakwe kwi-37 ° C kwi-90 min.Iziqwenga zejeli zaye zatshintshelwa kwiqula elitsha eliqulathe i-20 mM Tris, i-pH 8, kunye ne-5 μM ThT kwipleyiti esezantsi engabopheleliyo engama-96 ecacileyo.Fumana i-fluorescence eluhlaza kunye nemifanekiso yentsimi eqaqambileyo kwi-20x / 0.4 yokukhulisa.I-ImageJ yayisetyenziselwa uhlalutyo lomfanekiso.
Isisombululo se-NMR spectra sifunyenwe kwi-310 K kwi-600 MHz Bruker Avance Neo spectrometer exhotyiswe nge-QCI Quadrupole Resonance Pulsed Gradient Field Cryoprobe (HFCN).Iisampulu ze-NMR eziqulethe i-10 mg / mL iprotheni ene-homogeneous ebhalwe nge-13C, i-15N, echithwe kwi-20 mM Tris-HCl (pH 8), 0.02% (w/v) NaN3, 5% DO (v/v), (n = 1) .Ukutshintshwa kweekhemikhali ze-NT2RepCT kwi-pH 6.7 zisetyenziselwa ukunika i-peak 23 kwi-spectrum ye-2D ye-15N-HSQC.
I-magic angle spinning solid NMR (MAS) i-spectra ye-13C, i-15N-eleyibhile i-hydrogels yarekhodwa kwi-Bruker Avance III ye-spectrometer ye-HD kwi-800 MHz ixhotyiswe nge-3.2 mm 13C / 15N {1H} i-electronless probe.Ubushushu besampuli bulawulwa kusetyenziswa umlambo wegesi oguquguqukayo kwi-277 K. I-dimensional dipole rotational resonance (DARR) 76 kunye ne-radio frequency reconnection (RFDR) i-77 spectra ifunyenwe kwi-MAS frequencies ye-12.5 kHz kunye ne-20 kHz, ngokulandelanayo.I-Cross polarization (CP) ukusuka kwi-1H ukuya kwi-13C yenziwa ngokusebenzisa i-ramp ye-linear ukusuka kwi-60.0 ukuya kwi-48.0 kHz kwi-1H, 61.3 / 71.6 kHz kwi-13C (kwi-12.5 / 20 kHz MAS) kunye nexesha lokudibanisa i-0.5-1 ms.I-Spinal6478 decoupling kwi-73.5 kHz isetyenziswe ngexesha lokuqokelela idatha.Ixesha lokufumana laliyi-10 milliseconds kwaye ukulibaziseka komjikelo kwakuyimizuzwana eyi-2.5.Ulungelelwaniso lwe-Cα / Cβ oludityanisiweyo olulodwa olujongwe kwi-spectra ye-RFDR lwabelwa ngokusekwe kwi-charactical residue-type shifts chemicals shifts kunye ne-multiple-linked correlations kwi-spectra ye-DARR.
Iziko ledatha leZipper79 (https://services.mbi.ucla.edu/zipperdb/) lisetyenziselwe ukuvavanya i-flutter tendencies kunye ne-Rosetta energy ye-NT, NTFlSp, kunye ne-NTMiSp.I-database ye-Zipper ibala i-Rosetta Energy80, edibanisa imisebenzi emininzi yamandla yamahhala ukulinganisa kunye nokuhlalutya isakhiwo seprotheni.Inqanaba lamandla -23 kcal / mol okanye ngaphantsi libonisa ukuthambekela okuphezulu kwi-fibrillate.Amandla asezantsi athetha ukuzinza kwee-β-strands ezimbini kwi-zipper conformation.Ukongezelela, i-algorithm ye-Waltz yayisetyenziselwa ukuqikelela imimandla ye-amyloidogenic kwi-NT, i-NTFlSp kunye ne-NTMiSp Ref.81. (https://waltz.switchlab.org/).
Isisombululo seprotheni ye-NT saxutywa ne-2-(N-morpholino) ethanesulfonic acid (MES) buffer kwi-pH 5.5 kunye ne-6.0 ukuthoba i-pH ukuya kwi-pH 6 kunye ne-7, ngokulandelanayo.I-protein concentration yokugqibela yayiyi-100 mg / ml.
Imilinganiselo yenziwa kwi-J-1500 CD spectrometer (JASCO, USA) isebenzisa i-cuvette ye-300 μL kunye nomzila we-optical we-0.1 cm.Iiprotheyini zixutywe kwi-10 μM (n = 1) kwi-20 mM ye-phosphate buffer (pH 8).Ukuhlalutya ukuzinza kweprotheyini phambi kwetyuwa, iiprotheyini zahlalutywa kwi-concentration efanayo (n = 1) kwi-20 mM phosphate buffer (pH 8) equkethe i-154 mM NaF okanye i-NaCl, ngokulandelanayo.Izikrini zokushisa zirekhodwa kwi-222 nm ukusuka kwi-25 ° C ukuya kwi-95 ° C kunye nesantya sokufudumala kwe-1 ° C / min.Umlinganiselo weeproteni ezigoqiweyo zabalwa kusetyenziswa ifomula (KDmeasure – KDfinal)/(KDstart – KDfinal).Ukongeza, ii-spectra ezintlanu zirekhodwe kwisampulu nganye ukusuka kwi-260 nm ukuya kwi-190 nm kwi-25 ° C kwaye emva kokufudumeza ukuya kuma-95 ° C.Iziboniso ezihlanu zenziwe i-avareji, zagudiswa kwaye zaguqulelwa kwi-molar ellipticity.Idatha yahlalutywa kusetyenziswa iPrism 9.
Ubunzulu be-fluorescence be-His-NT-GFP (300 mg/mL, 80 µL) bulinganiswe nge-triplicate (n = 3) kwiipleyiti zeCorning ezingama-96 ezinomzantsi okhanyelayo omnyama (Corning Glass 3881, USA) phantsi kweemeko ezimileyo.Ukulinganisa iisampulu nge-fluorescence-based plate reader ene-excitation wavelength ye-395 nm kwaye urekhode ukukhutshwa kwi-509 nm phambi kwe-gelation kunye neeyure ezi-2 kamva kwi-37 °C.Idatha yahlalutywa ngePrism 9.
I-Purine nucleoside phosphorylase ikiti yokuvavanya umsebenzi (indlela ye-fluorometric, uSigma Aldrich) isetyenziswe ngokwemiyalelo yomenzi.Ukulinganisa umsebenzi kwiigels kunye nezisombululo eziqulethe i-Hi-NT-PNP, xuba i-10 ng ye-His-NT-PNP kunye ne-100 mg / mL NT kumthamo opheleleyo we-2 µL kuba ijeli inike umqondiso ngaphezu kwexesha lokufumanisa isethi.Ulawulo lweejeli kunye nezisombululo ngaphandle kwe-His-NT-PNP zabandakanywa.Imilinganiselo yenziwe kabini (n = 2).Emva kokuba umsebenzi ulinganisiwe, umxube wokuphendula ususiwe kwaye i-gel ifotwe ukuqinisekisa ukuba i-gel ihleli ilungile ngexesha lokulinganisa.Idatha yahlalutywa kusetyenziswa iPrism 9.
Ngolwazi oluthe kratya malunga noyilo lwesifundo, jonga i-Nature study abstract eqhagamshelwe kweli nqaku.
Amanani 1 kunye ne-2 abonisa idatha yokuqala.1c, 2a–c, 3a, b, e–g, 4, 5b, d, f, kunye no-6, amaFigs awongezelelweyo.3, umkhiwane owongezelelweyo.5a, d, ikhiwane elongezelelweyo.6 kunye nekhiwane elongezelelweyo.8. Idatha yedatha esuka kolu phononongo ibanjwe kwiziko ledatha leZenodo https://doi.org/10.5281/zenodo.6683653.Idatha ye-NMR efunyenwe kolu phononongo yathunyelwa kwindawo yokugcina i-BMRBig phantsi kwe-ID yokungena bmrbig36.Izakhiwo ze-GFP kunye ne-PNP zithathwe kwi-PDB (GFP 2B3Q, PNP 4RJ2).
Rising, A. kunye noJohansson, J. Ukusonta isilika yesigcawu eyenziweyo.Imichiza yeSizwe.ibhayoloji.11, 309-315 (2015).
Babb, PL et al.I-Nephila clavipes genome ibalaselisa ukungafani kofuzo lwesilika yesigcawu kunye nembonakalo yazo entsonkothileyo.IGenette yeSizwe.49, 895-903 (2017).
Ixesha lokuposa: Mar-12-2023