310 10*1mm Kuleana kila coiled tubing chemical component, ʻO nā kikowaena N-terminal o spdroin hoʻokumu i nā hydrogels e pili ana i nā fibrils amyloid a hāʻawi i kahi kahua no ka immobilization protein.

Mahalo no kou kipa ʻana iā Nature.com.Ke hoʻohana nei ʻoe i kahi polokalamu kele pūnaewele me ke kākoʻo CSS palena ʻole.No ka ʻike maikaʻi loa, manaʻo mākou e hoʻohana i kahi polokalamu kele pūnaewele hou (a i ʻole e hoʻopau i ke ʻano Compatibility Mode ma Internet Explorer).Eia kekahi, e hōʻoia i ke kākoʻo mau, hōʻike mākou i ka pūnaewele me ka ʻole o nā styles a me JavaScript.
ʻO nā slider e hōʻike ana i ʻekolu ʻatikala ma kēlā me kēia paheʻe.E hoʻohana i nā pihi hope a i hope no ka neʻe ʻana i nā paheʻe, a i ʻole nā ​​pihi hoʻokele paheʻe ma ka hopena e neʻe i kēlā me kēia paheʻe.

Hōʻike

310 10*1mm Mea kūʻai aku i nā mea hoʻolako i nā mea kūʻai aku

Hoʻohui Kimia

Na Waiwai Mechanical

ʻO nā protein siliki spider recombinant (spider silk proteins) he nui nā mea hiki ke hoʻohana i ka hoʻomohala ʻana i nā biomaterial hou, akā ʻo ko lākou ʻano multimodal a me ka hoʻohui ʻana he mea paʻakikī ke loaʻa a maʻalahi hoʻi e hoʻohana.Ma ʻaneʻi, hōʻike mākou i nā protein spidroin liʻiliʻi recombinant a, ʻo ka mea nui, ʻo ka N-terminal domain (NT) ponoʻī e hana wikiwiki i nā hydrogels kākoʻo ponoʻī a me ka maopopo ma 37 °C.ʻO nā protein hui pū ʻia me ka NT a me ka ʻōmaʻomaʻo fluorescent protein a i ʻole ka purine nucleoside phosphorylase e hana pono i nā protein fusion holoʻokoʻa.Hydrogels.Hōʻike kā mākou mau hopena i ka recombinant NT a me nā protein fusion e hāʻawi i nā hua hōʻike kiʻekiʻe a hāʻawi i nā hydrogels me nā waiwai hoihoi e like me ka transparency, gelation me ka ʻole crosslinking, a me ka immobilization pololei o nā protein ikaika ma ke kiʻekiʻe kiʻekiʻe.
Loaʻa i nā pūnawelewele he ʻehiku mau pūʻulu siliki like ʻole, e hana ana kēlā me kēia i kahi ʻano siliki kikoʻī.ʻO nā ʻano silika ʻehiku i haku ʻia me nā protein siliki spider (spidroins) ma kahi o 6000 koena ka lōʻihi a loaʻa kahi ʻāpana kikowaena nui i hoʻopuni ʻia e nā kikowaena spherical N- a me C-terminal (NT a me CT)1,2.ʻO ke ʻano siliki i aʻo nui ʻia, ʻo ka ampulla mua, ua hana ʻia e ka puʻu ampulla mua.I loko o kēia gland, kahi monolayer o nā epithelial cell synthesizes spidroin proteins a hūnā iā lākou i loko o ka lumen o ka gland, kahi i loaʻa i kahi ʻano hoʻoheheʻe (doping) i nā kiʻekiʻe kiʻekiʻe (30-50% w / v)3,4.Ua hoʻopaʻapaʻa ʻia ka hoʻonohonoho ʻana a me ka hoʻohālikelike ʻana o nā protein ampullar spidroin nui i loko o ka gland, akā ʻo ka hapa nui o nā hōʻike hoʻokolohua e hōʻike ana i ka hele ʻana o kahi conformation helical maʻamau a / a i ʻole nā ​​​​micellar a lamellar structures5,6,7,8,9,10.ʻOiai nā kāʻei kapu repetitive e hoʻoponopono i nā ʻano mechanical o nā fiber silk, e hana ana i nā nanocrystals β-sheet a me nā hale amorphous11,12,13,14,15, hoʻoponopono nā kikowaena hope i nā fiber silk i pane i ka hoʻololi ʻana i nā kūlana ma ka gland silika16,17,18.Ma o ka hoomalu ana i ka hana silika, 19. Ua hoomaluia na kikowaena kikowaena a he mea ma'amau paha ko lakou hana i na protein spidrin a pau 2,20,21.I ka wā e hele ai i loko o ka gland, emi ka pH o ka spdroin mai kahi o 7.6 a i <5.716 a hoʻonui me ka ʻoki a me ka hoʻolōʻihi ʻia e ka neʻe ʻana ma o ke aʻa haʻahaʻa haʻahaʻa.I ka hoʻonā, CT he α-helical constitutive parallel dimer17, akā i ka pane ʻana i ka pH haʻahaʻa a me nā ikaika shear, wehe ʻo CT a hoʻololi i nā β-layers16, 17, hiki paha ke hoʻoulu i nā β-layers i nā ʻāpana repetitive o Convert 16. NT he monomeric ma lalo o ʻO nā kūlana e hōʻike ana i nā kūlana i loko o ka lumen o ka gland a me ka hoʻopili ʻana i ka solubility o ka spidroin, akā i ka hoʻohaʻahaʻa ʻana i ka pH, ka protonation o ka nui o nā kaulahao ʻaoʻao carboxylic e alakaʻi i ka dimerization o NT me kahi pKa ma kahi o 6.5, a laila hoʻopaʻa i ka NT a hoʻopaʻa i ka spidroin i ka nui. ka nui.pūnaewele16,18.No laila, he koʻikoʻi ko NT i ka hoʻokumu ʻana i ka filament, e hoʻololi ana mai kahi monomer i ka uhi ʻana i kahi dimer i ka fiber23,24,25.Noho ʻo NT i mea hiki ke hoʻoheheʻe ʻia a helical ma lalo o nā kūlana a pau i aʻo ʻia a hiki i kēia lā16, 18, 19, 20, 26, 27, 28, 29, ka mea i hoʻoulu i kona hoʻomohala ʻana ma ke ʻano he lepili hoʻonui solubility no ka hana ʻana i nā protein heterologous.
ʻO ka pūmua silika spider mini recombinant, he hoʻokahi NT, hoʻokahi wahi pōkole pōkole, hoʻokahi CT, a me kahi hōʻailona His6 (His-NT2RepCT) no ka hoʻomaʻemaʻe ʻana, hiki ke hoʻoheheʻe ʻia i loko o ka paʻa wai e like me ka protein silk spider maoli a hoʻohālike i nā ʻano koʻikoʻi maoli o ka spider silika. .uhi 25.31.Hiki ke wili ʻia kāna-NT2RepCT i loko o nā fiber mau me ka mīkini biomimetic kahi i hoʻokuʻu ʻia ai ka uhi ʻana o ka pH 8 i loko o ka ʻauʻau wai pH 525,32,33,34,35.ʻO ka bioreactor fermentation o E. coli e hōʻike ana i kāna-NT2RepCT a ma hope o ka mālama ʻana ma hope o ka hopena ma hope o ka 14 g/L hua ma hope o ka hoʻomaʻemaʻe ʻana.ʻO ka hua kiʻekiʻe, ka solubility kiʻekiʻe, a me ka pane kūpono o kāna-NT2RepCT i nā kūlana acidic e pili ana iā NT23, 25, 34.
Ma ʻaneʻi, hōʻike mākou i ka hoʻokumu wikiwiki ʻana o nā hydrogels akaka mai nā protein spidroin recombinant, me ka NT wale nō, ma ka hoʻoulu ʻana i kahi solution protein ma 37 °C.Ke hoʻohana nei i ka thioflavin T fluorescence (ThT), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR) a me transmission electron microscopy (TEM), ua ʻike mākou ua hoʻololi ʻia ka NT a me ka microspider proteins i nā β-sheets a me nā fibrils like amyloid. ke hana ʻia nā gels.Eia kekahi, ʻo nā protein fusion o NT a me ka ʻōmaʻomaʻo fluorescent protein (GFP) a i ʻole purine nucleoside phosphorylase (PNP) e hana i nā hydrogels me nā ʻāpana fusion holoʻokoʻa.ʻO ka hōʻike kiʻekiʻe-throughput i loko o nā pūʻali heterologous, i hui pū ʻia me ka hoʻokumu wikiwiki ʻana o nā hydrogels ma lalo o nā kūlana physiological, wehe i ka hiki ke hana i nā hydrogels me nā hana ʻenehana.
ʻAʻole like me ka hapa nui o nā protein spidroin recombinant36 i hōʻike ʻia, paʻa ʻo His-NT2RepCT ma Tris-HCl buffer ma ka pH 8 a hiki ke hoʻopaʻa ʻia a hiki i 500 mg/mL me ka ʻole o ka ua25.No laila, kāhāhā mākou i ka ʻike ʻana i ka hana wikiwiki ʻana o kēia pūmua i nā hydrogels ponoʻī i ka wā i hoʻomoʻa ʻia ma 37 ° C (Fig. 1b-d).Ua hōʻike ʻia nā haʻawina hou aʻe ua loaʻa ʻo His-NT2RepCT gelation ma luna o kahi ākea o ka nui o ka protein (10-300 mg / mL) a ua hoʻopili ʻia kēia ʻano me ka manawa gelation (Fig. 1c a me Supplementary Fig. 1).No ka ʻike ʻana i nā ʻāpana o kāna-NT2RepCT e hoʻokumu i ka hydrogel, a laila nānā mākou i kēlā me kēia kikowaena i kēlā me kēia ʻano a me nā hui like ʻole me ka hoʻohana ʻana i kahi flask inversion assay (Figure 1a, b).ʻO nā hakina a pau i hoʻāʻo ʻia o ka spidroin recombinant i hana i nā gels (ma kahi ʻano protein o 300 mg / mL) ma lalo o 1 h, koe wale no ka precipitated 2Rep (Fig. 1b).Hōʻike kēia i ka NT a me CT wale nō, i hui pū ʻia, a i pili pū me ka hana hou ʻana, hiki ke gel ma 37 ° C a ʻaʻole pili ka His6 tag i kēia kaʻina hana i kekahi ʻano nui.Hāʻawi ʻia i ka manaʻo maʻamau ʻo NT he protein paʻa loa a paʻa, a ʻo nā hōʻike mua o ka recombinant spidroin hydrogels ua hoʻopiʻi i nā hopena gelation i nā loli conformational i nā wahi hou a / a i ʻole CT, hiki iā NT ponoʻī.ʻAʻole i manaʻo ʻia ka loaʻa ʻana o ka gelation.Pākuʻi Pākuʻi 1) 37, 38, 39. ʻO ka mea kupaianaha, ua hoʻopili ʻia ʻo NT i loko o 10 mau minuke ma kahi o ≥ 300 mg / mL (Fig. 1c).Hōʻike ʻia nā hoʻokolohua hoʻololi vial me nā ʻano ʻokoʻa o ka NT ma> 50 mg/mL ua ʻoi aku ka wikiwiki o ka hoʻonā NT ma mua o kāna-NT2RepCT ma ke ʻano kūlike (w / v, Kiʻi 1c).
Hōʻike schematic o nā ʻano hana spidrin i aʻo ʻia ma kēia hana.b Ka manawa gel ma 37 °C no nā ʻano protein spidrin recombinant (300 mg/mL) i hōʻoia ʻia ma ka hoʻohuli ʻana i ka hue.CT gel koke me ka incubation (<300 mg/mL), 2Rep precipitates (300 mg/mL, 5 mm scale).c Ka manawa Gel o kāna-NT2RepCT a me NT ma ka hōʻike ʻia ʻana o ka protein ma 37°C.d Nā kiʻi o kāna-NT2RepCT a me NT hydrogels me ka spider a me ka huapalapala "NT" i paʻi ʻia ma lalo, paʻa (ʻelua 200 mg/mL, pae pālākiō 5 mm).
He ʻokoʻa nā kala like ʻole nā ​​hydrogels i hana ʻia e nā protein spidroin recombinant like ʻole, a ʻo ka nānā ʻana i ka maka ʻōlohelohe e hōʻike ana i nā degere like ʻole o ke aniani (Fig. 1b).Maikaʻi loa nā gels NT a lilo nā gels ʻē aʻe i opaque.ʻO kāna-NT2RepCT a me NT gels i hoʻolei ʻia i loko o nā paipu cylindrical hiki ke hoʻoneʻe ʻia mai ka mold paʻa (Fig. 1d).
No ka hoʻāʻo ʻana inā he gel kilika kilika maoli ma lalo o nā kūlana i loaʻa i kēia manawa ke kumu o ka gelation o nā protein spidroin recombinant, ua hōʻiliʻili ʻia nā uhi mai ka ampulla gland nui o ka spider bridge Swedish (Larinioides sclopetarius).Ua mālama ʻia nā pale ma 20 mM Tris-HCl buffer ma 50 mg / mL (ma muli o ke ana ʻana i ke kaumaha maloʻo), akā ʻaʻole i ʻike ʻia ka gelation i ka wā o ka incubation 21 lā ma 37 ° C (Supplementary Figure 2a).
No ka helu ʻana i kēia mau gels, hiki ke hoʻohana ʻia nā ana rheological no ke aʻo ʻana i ke kaʻina hana gelation a hoʻoholo i nā waiwai mechanical āpau.ʻO ka nānā ʻana i ka modulus mālama (elasticity) i nā mahana kiʻekiʻe e hiki ke hāʻawi i ka ʻike e pili ana i ka wela gelling a me nā waiwai viscoelastic o ka uhi.ʻO nā hoʻokolohua piʻi wela (e hoʻohana ana i ka 1 ° C / min ma 25-45 ° C, e pili ana i nā haʻawina mua e hoʻohana ana i nā ʻenehana siliki kūlohelohe) 40,41 i hōʻike i ka hoʻonui ʻana o ka moduli mālama o kāna-NT2RepCT a me NT me ka piʻi ʻana o ka mahana.ua hoʻonui ʻia (Fig. 2 a me ka Fig. 3 Hoʻohui).ʻO ka mea nui, ua hoʻomaka ka ulu ʻana o ka module NT ma kahi haʻahaʻa haʻahaʻa i hoʻohālikelike ʻia me kāna-NT2RepCT, e kūlike me ka manawa gel wikiwiki i ʻike ʻia i ka wā i hoʻopili pololei ʻia ai ʻo NT me kāna-NT2RepCT ma 37 ° C (Figure 1).Ma hope o ka hāʻule ʻana o ka mahana, ʻaʻole i hoʻi ka modulus mālama i nā waiwai haʻahaʻa a noho mau ma luna o ka modulus nalowale (e nānā i ke kiʻi hou.Ma hope o ka gelation, ʻo ka modulus elastic hope loa mai ka 15 a hiki i 330 kPa no kāna-NT2RepCT hydrogels ma kahi ʻano o 100-500 mg / mL, a ʻo ka modulus elastic hope loa no NT hydrogels (100-500 mg / mL) mai 2 a 1400. kPa (Fig. 2 a me ka ʻikepili ramp piha) e ʻike i ka Fig 3.
a Hoʻololi i ka mahana i nā ana o kāna-NT2RepCT (300 mg/mL) a me b NT (300 mg/mL) me ka haʻalulu.Hōʻike nā pua i ke ʻano o ka mahana, a ʻo ka ʻulaʻula māmā o ka ʻikepili waihona waihona e hōʻike ana i ka hoʻāʻo ʻana i nā koina torque haʻahaʻa no ka mea kani ma mua o ka mea i kuhikuhi ʻia e ka mea hana, ʻo ia ke kumu o ka piʻi ʻana o ka walaʻau.c Hoʻopili hope-module o kāna-NT2RepCT a me NT ma hope o ka wela kiʻekiʻe (100, 300, a me 500 mg / mL).Lawe ʻia nā heluhelu module āpau ma ke alapine o 0.1 Hz.
Ma ke ʻano he ala kūpono no ka noiʻi ʻana i nā loli conformational e pili ana me ka gelation, ua hoʻopaʻa mākou i ka FTIR spectra o kāna-NT2RepCT a me NT ma mua a ma hope o ka gelation ma 37 ° C (Figure 3a,b).E like me ka mea i manaʻo ʻia, pili ka spectra o kāna-NT2RepCT a me NT solutions i nā protein e hōʻike ana i ka α-helix/random coil second structure, me kahi hui i haʻi ʻia ma 1645 cm-1.No nā hydrogels ʻelua, ʻo ka gelation ka hopena i ka hoʻokumu ʻia ʻana o nā lima ʻelua ma ka waena I band ma kahi o 1617 cm-1 a me 1695 cm-1 (Fig. 3a, b), e hōʻike ana i ke kūkulu ʻia ʻana o nā hale β-sheet antiparallel.Hiki ke ʻike maopopo ʻia kēia mau hoʻololi i ka lua o ka derivative a me ka ʻokoʻa gelation spectra (Supplementary Fig. 4b).ʻO nā pūʻulu ʻelua o ka NT β-layer ua ʻoi aku ka nui ma mua o kāna-NT2RepCT, e hōʻike ana ua ʻoi aku ka nui o ka nui o nā ʻāpana β-layer i ka NT hydrogel ma mua o ka hydrogel NT2RepCT.
he FTIR absorption spectra o His-NT2RepCT a me b NT (ʻelua 500 mg/mL) ma mua (solution) a ma hope o (gel) incubation ma 37°C.c kiʻi TEM o 50 mg/ml NT2RepCT gels a me d NT.ʻO ka pā unahi 200 nm.e Nā anawaena fiber o kāna-NT2RepCT a me NT hydrogels.n = 100 ana fibrils, p <0.0001.Hōʻike nā pahu kuhi i ka ʻokoʻa maʻamau.ʻO ke kikowaena o nā pahu hewa ka mean.Ua hoʻohana ʻia kahi hoʻāʻo t i hoʻohui ʻole ʻia (ʻelua huelo) no ka nānā ʻana i nā helu.f ThT fluorescence o nā ʻano protein spidroin recombinant (100 mg/mL) ma 37 °C me ka lulu ʻole.g NT (100 mg / mL) hoʻokolohua inoculation mai 100 mg / mL NT gel me 0%, 5%, 10%, a me 20% mau hua.
ʻO ka nānā ʻana o ka gel me ka hoʻohana ʻana i ka microscopy electron transmission (TEM) i hōʻike ʻia aia ka hydrogel i nā fibrils like amyloid (Fig. 3c, 3d).Ua hoʻolōʻihi ʻia nā fibrils i hana ʻia e NT (5-12 nm ke anawaena) a ʻaʻohe lālā, ʻoiai ʻo kāna-NT2RepCT fibrils he pōkole ka lōʻihi a ʻoi aku ka nui o ke anawaena (7-16 nm) (Fig. 3e).Ua ʻae kēia mau hopena iā mākou e hahai i nā kinetics o fibrosis me ka hoʻohana ʻana i ka thioflavin T (ThT) assay.No nā protein spidroin recombinant a pau, ua hoʻonui ʻia ka hōʻailona fluorescent i ka wā i hoʻomoʻa ʻia ai nā laʻana ma 37 °C (Fig. 3f, Supplementary Fig. 5a).E like me kēia ʻike, ʻike ʻia ka hoʻokolohua microscopic o NT a me kāna-NT2RepCT ma lalo o nā kūlana gelling i hōʻike ʻia i ka piʻi ʻana o ka fluorescence ThT me ka ʻike ʻole ʻia o ka hōʻiliʻili kūloko o ThT-positive aggregates (Supplementary Fig. 5b, c).ʻAʻole i hui pū ʻia ka hoʻokumu ʻana o nā fibrils ThT-positive me ka piʻi ʻana o ka NT a me kāna-NTCT turbidity (Supplementary Fig. 5d), ʻo ia hoʻi e hiki ke hana i kahi pūnaewele o nā fibrils i ka gel me ka ʻole o ka hoʻohālikelike ʻana i ka mālamalama gel.ʻO ka lūlū ʻana ma ka hoʻohui ʻana i nā liʻiliʻi liʻiliʻi o nā fibrils i hana mua ʻia e hiki ke hoʻoikaika nui i ka hoʻokumu ʻana o ka fibril o kekahi mau amyloids42,43,44 akā hoʻohui i 5%, 10% a i ʻole 20% (w/w) NT i kahi hopena o NT hydrocoagulants.hua hua (Fig. 3g).Malia paha ma muli o ka paʻa ʻana o nā fibrils i ka hydrogel a ʻaʻole hiki ke hoʻohana ʻia e like me nā hua.
ʻO ka hana i manaʻo ʻole ʻia o nā protein spidroin recombinant i nā wela kiʻekiʻe ua hoʻoikaika hou i nā haʻawina spectroscopy nuclear magnetic resonance (NMR) e ʻike i nā loli conformational e pili ana i ka hoʻokumu ʻana i ka gel.NMR spectra of His-NT2RepCT solutions recorded over time at 37°C ua hōʻike i ka CT i ʻāpana ʻāpana, ʻoiai ua nalowale nā ​​hōʻailona NT a me 2Rep (Fig. 4a), e hōʻike ana ʻo ia ka nui o NT a me 2Rep i hoʻonohonoho ʻia i ka hoʻokumu ʻana o His- NT2RepCT hydrogel.Ua hoʻemi ʻia ka hōʻailona CT i ka 20% o kona ikaika mua, e hōʻike ana ua hoʻopaʻa ʻia ʻo CT a hoʻokomo ʻia i loko o ka hale hydrogel.No kahi ʻāpana liʻiliʻi o ka CT, e like me ka mobile e like me ka preincubated sample a no laila ʻike ʻia e ka solution NMR, nele ka spectra i nā hōʻailona no nā koena i kūkulu ʻia he 10 mua, ma muli paha o ka immobilization paʻakikī o ka ʻāpana pili o kāna-NT2Rep.ʻO ka NMR spectra o ka -state of hydrogels -NT2RepCT i hōʻike i ka nui o nā α-helice a me nā β-layers a, i ka liʻiliʻi liʻiliʻi, ka conformation coil random (Fig. 4b).Ua hōʻike ʻia ka hoʻololi ʻana o ka hoʻololi kemika o nā koena methionine ma NT wale nō ua hoʻololi ʻia kēia kikowaena i kahi ʻano β-pepa.Ua hōʻike ka spectra pili manawa o NT i ka hoʻonā i ka emi ʻana o ka hōʻailona hōʻailona (Fig. 4c), a me ka NMR solid-state o NT hydrogels i hōʻike i ka hapa nui o nā koena NT i hoʻololi ʻia i nā hale β-sheet (Fig. 4d).ʻAʻole hiki ke hoʻoholo ʻokoʻa ʻia ka conformation o 2Rep ma muli o kona makemake e hōʻuluʻulu.Eia naʻe, ʻano like loa ke ʻano o ka NMR spectra o ka NTCT a me kāna-NT2RepCT hydrogels (Fig. 4b; Fig.No ka CT hydrogels, α-helices, β-sheets, a me nā hale kiʻekiʻe helical maʻamau i loaʻa i ke ola (Supplementary Fig. 6d).Hōʻike kēia i kekahi mau ʻāpana o ka CT e noho α-helice a ʻo kekahi e lilo i β-pepa.No laila, hōʻike ʻia nā hopena o ka spectroscopy NMR he mea nui ka NT no ka hoʻokumu ʻana i ka hydrogel a hoʻololi pū i kahi conformation β-sheet ma ka hui ʻana me 2Rep a me CT.E like me kēia, ua ʻike mākou i kēia manawa ua hoʻokumu ʻia nā zippers spatial amyloid i nā helices ʻelima a pau o ka domain NT, a ua wānana ka Waltz algorithm i kahi ʻāpana amyloidogenic i ka helix 1 (Fig. 4e).
2D spectra o 15N-HSQC 10 mg/mL His-NT2RepCT solution ma mua o (uliuli) a me 19 mau hola ma hope o ka hoʻoulu ʻana (ʻulaʻula) ma 37°C.Hōʻike ʻia nā piko keʻa pākahi i ka spectrum ʻulaʻula a me F24, G136, polyA i ka spectrum polū e nā hōʻailona amino acid a me nā helu koena.Hōʻike nā mea hoʻokomo i ka hilinaʻi o ka ikaika o ka hōʻailona i ka manawa no nā koena i koho ʻia mai nā kikowaena NT, 2Rep, a me CT.b ʻO ka lāʻau radiofrequency solid-state (RFDR) o kāna-NT2RepCT hydrogels.Ua hoʻoholo ʻia ka hoʻoponopono ʻana o nā koena Cα/Cβ i ka spectra RFDR e ka hoʻohālikelike ʻana me nā hoʻololi kemika peptide model a me nā waiwai i loaʻa mai nā statistics82,83 a me kā lākou mau hale kiʻekiʻe.SSB – ʻaoʻao ʻaoʻao ʻaoʻao.c Hoʻokahi-dimensional spectra o 15N-HSQC 10 mg/mL NT solution i ka hoʻoulu ʻana ma 37 °C no 36 mau hola.Hōʻike ka inset i ka ikaika volumetric me ka manawa.d Kūlana paʻa RFDR spectra o NT hydrogels.Hōʻike ʻia nā correlations o nā koena Cα / Cβ a me ko lākou mau hale kiʻekiʻe i ʻike ʻia ma ka spectra RFDR.e Ma muli o ka NT45.79 fibrillation propensity profile mai ka waihona Zipper (https://services.mbi.ucla.edu/zipperdb/).Hōʻike ʻia ka ikehu Rosetta o ka puka aniani uila uila o ka hexapeptide i kcal/mol.Hōʻike nā pā ʻulaʻula i nā hexapeptides me kahi kiʻekiʻe fibrosis propensity (Rosetta ikehu ma lalo o -23 kcal/mol; ma lalo o ka laina kiko).Hōʻike nā ʻōmaʻomaʻo i nā ʻāpana me ka ikaika Rosetta ma luna o ka paepae a no laila ʻaʻole hiki ke hana i nā zippers steric.Ua kāpae ʻia nā ʻāpana i loaʻa ka proline mai ka nānā ʻana (me ka ʻole o nā kolamu).Hōʻike nā huinahā i nā wahi o ka amyloidosis i wānana ʻia e ka Waltz algorithm81 (https://waltz.switchlab.org).Aia ke kaʻina o nā koena amino acid o NT ma luna, a ʻo nā ʻano o nā koena i loaʻa i loko o ka hale kiʻekiʻe β (i hoʻoholo ʻia e ka solid-state NMR spectroscopy) e hōʻike ʻia i ka ʻulaʻula.Ua koho ʻia nā kūlana o nā ʻelima NT α-helice e like me (H1-H5)28.
Ma ka pH <6.5, emi mai ka HT, ke kū'ē i ka denaturation i hoʻokomo ʻia i ka wela a i ʻole ka urea18.No ka wehewehe ʻana i ke ʻano o ka dimerization a me ke kūpaʻa o NT i ka gelation, ua hoʻomalu ʻia nā hopena i loaʻa ka 100 mg/ml NT ma ka pH 8, 7, a me 6 me ka hoʻohana ʻana i ka hoʻāʻo inversion vial.Hoʻokomo ʻia nā laʻana NT ma ka pH 8 a me 7 gelled ma hope o 30 min ma 37 ° C, akā ua mau ka pH 8 gel, ʻoiai ʻo ka pH 7 gel i hōʻike i kahi precipitate ʻike ʻia (Fig. 5a).I ka hoʻokaʻawale ʻana, ʻaʻole i hana ʻia kahi hoʻonā i loaʻa ka HT ma ka pH 6 i gel, a hiki ke ʻike ʻia kahi wai nui ma hope o 20 min ma 37 ° C.Hōʻike kēia i nā dimer iā lākou iho a / a i ʻole ko lākou kūpaʻa kiʻekiʻe i hoʻohālikelike ʻia me nā monomers e pale i ka gelation.ʻAʻole i manaʻo ʻia ka hoʻokumu ʻana o kahi precipitate no NT ma ka pH 7 a me 6, no ka mea, ua hōʻike ʻia ua hiki ke hoʻoheheʻe ʻia ka NT ma 200 mg/ml27, maʻalahi e hoʻihoʻi hou ma hope o ka denaturation wela, a mālama pū kekahi i kahi α-helix ma nā haʻahaʻa haʻahaʻa. pH 18. ʻO kahi wehewehe kūpono no kēia mau ʻokoʻa, ʻo ia ka mea i hana ʻia nā hoʻokolohua i hōʻike mua ʻia ma ka lumi wela a i ʻole ma lalo, a i ʻole ma kahi haʻahaʻa haʻahaʻa o ka protein16,18,19.
NT vial inversion ho'āʻo (100 mg/mL) ma pH 8, 7, 6 a me 154 mM NaCl (pH 8) ma hope o incubation ma 37°C.b NT CD spectra me 154 mM NaF a me 154 mM NaCl, pakahi.Hoʻololi ʻia ka ellipticity molar ma 222 nm i ka hapa o nā ʻōpala maoli.c NT inversion assay (100 mg/mL) NT* (37 °C a me 60 °C), NTA72R (37 °C), a me kāna-NT-L6 (37 °C a me 60 °C).d CD spectra o NT mutants NT*, NTA72R, a me His-NT-L6.Hoʻololi ʻia ka ellipticity molar ma 222 nm i ka hapa o nā ʻōpala maoli.e ho'āʻo hoʻohuli ʻia o NTFlSp, NTMiSp a me NTMiSp i hōʻemi ʻia (100 mg/mL).ʻO ka pā unahi 5 mm.f CD spectra o NT, NTFlSp, NTMiSp a hoemi NTMiSp.Hoʻololi ʻia ka ellipticity molar ma 222 nm i ka hapa o nā ʻōpala maoli.Hōʻike ʻia nā kikoʻī NT piha ma 25 °C a me 95 °C ma ke Kiʻi 8.
Hoʻoholo ka paʻakai physiological i nā pilina electrostatic ma waena o nā subunits NT a me ka dimerization o ka hoʻololi ʻana o NT i ka pH18 haʻahaʻa.Ua ʻike mākou ʻo ka hiki ʻana mai o 154 mM NaCl a me NaF i kaohi maoli i ka gelation, i kēlā me kēia (Fig. 5a, b; Hoʻohui Fig. 2b) a ua hoʻonui kēia mau paʻakai i ka paʻa wela o nā monomers NT (Fig. 5b, Fig 8). .Hōʻike pū ia i ka hoʻonui ʻana i ka paʻa, ma mua o ka dimerization, e pale i ka hoʻokumu ʻana o ka gel.
No ka ʻimi hou ʻana i ke kuleana o ka dimerization protein a me ke kūpaʻa i ka gelation, ua hoʻohana mākou i ʻelua mau mutants, NT* a me NTA72R, e noho mau ana ka monomeric ma ka pH28.30 haʻahaʻa.ʻO NT* he mutant hoʻihoʻi pālua i hoʻopalahalaha ʻia ka puʻunaue dipolar o ka monomer, e pale ana i ka dimerization a hoʻonui nui i ka paʻa monomer.ʻO NTA72R kahi dipole i hoʻopiʻi ʻia, akā aia ʻo Ala i pani ʻia ʻo Arg ma ka palena dimer, no laila ke keʻakeʻa nei nā hoʻololi i nā pilina subunit e pono ai no ka dimerization.Ma ka incubation ma 37 ° C, NT * ʻaʻole i hana i kahi hydrogel, aʻo NTA72R i hana i kahi gel opaque no 15 min (Fig. 5c).No ka mea, ʻaʻole hiki i ka NT* a me ka NTA72R ke dimerize akā ʻokoʻa i ka paʻa monomer (Fig. 5d), hōʻike ikaika kēia mau hopena i ka pale ʻana o ka thermodynamic kiʻekiʻe i ka NT mai ka gelling.Kākoʻo pū ʻia kēia e ka hana ʻana o ka HT * i ka gel i ka wā paʻa ʻole i ka wela kiʻekiʻe (ma hope o 8 min ma 60 ° C; Fig. 5c).Ua hōʻike mua ʻia ʻo ke kiʻekiʻe o ka methionine ma NT e hoʻoheheʻe i kona pelu maoli ʻana a ʻeono Met i Leu pani (i ʻōlelo ʻia ma aneʻi ʻo His-NT-L6) e hoʻopaʻa ikaika i ka monomer NT46.Ma muli o ka manaʻo e koi ʻia ka hoʻololi ʻana no ka hoʻokumu ʻana i ka gel NT, ua ʻike mākou ʻaʻole i gel ka His-NT-L6 stable mutant ma 37 ° C (Figure 5c, d).Eia naʻe, ua hana pū ʻo His-NT-L6 i kahi gel ma ka incubation ma 60 ° C no 60 min (Fig. 5c).
ʻO ka hiki o NT ke hoʻololi i nā hale β-pepa a hoʻokumu i nā hydrogels e pili ana i kekahi akā ʻaʻole nā ​​​​kaulana NT āpau o spdroin.ʻO nā NT mai nā ʻano siliki like ʻole a me nā ʻano spider, Trichonephila clavipes (NTFlSp), i hoʻokumu i nā gel me ka haʻahaʻa haʻahaʻa o ka methionine a me ke kūpaʻa wela kiʻekiʻe (Fig. 5e, f a me ka Papa Hoʻohui 2).ʻO ka hoʻohālikelike, NT mai ka spidroin protein ampullar liʻiliʻi mai Araneus ventricosus (NTMiSp) me ke kūpaʻa haʻahaʻa haʻahaʻa a me ka maʻi methionine kiʻekiʻe ʻaʻole i hana i nā hydrogels (Supplementary Table 2 a me Fig. 5e, f).Hiki ke pili i ka hope me ka loaʻa ʻana o nā paʻa disulfide intramolecular29,47.I ka manawa like, i ka wā i hoʻemi ʻia ai nā paʻa disulfide o NTMiSp, ua hana ia i kahi hydrogel ma hope o ka incubation ma 37 ° C no 10 min (Fig. 5e).I ka hopena, pono e hoʻomaopopo ʻia he mea koʻikoʻi ka flexibility structural, akā ʻaʻole ʻo ia wale nō, criterion no ka hoʻokumu ʻana i kahi gel mai NT.ʻO kekahi kumu ʻē aʻe e pili ana i ka makemake e hana i nā fibrils amyloid, a me ka nānā ʻana me ka waihona zipper a me ka Waltz algorithm i hōʻike i ka pilina ma waena o ka hiki ke hana i nā gels a me ka hiki ʻana o nā ʻāpana amyloidogenic, a me ka nui o nā wahi i wānana ʻia. e hana i nā zippers steric.Loaʻa kahi pilina (Supplementary Table 2 and Supplementary Fig. 9).
ʻO ka hiki i ka NT ke hana i nā fibrils a hana i nā gels ma lalo o nā kūlana maikaʻi i alakaʻi iā mākou i ka hypothesize e hiki i ka hui ʻana o NT me nā ʻāpana protein ʻē aʻe ke hana i nā gels me ka hana piha o nā hoa hui.No ka hoʻāʻo ʻana i kēia, ua hoʻokomo mākou i ka protein fluorescent green (GFP) a me ka purine nucleoside phosphorylase (PNP) ma ka C-terminus o ka NT, kēlā me kēia.Ua hōʻike ʻia nā protein fusion i loaʻa i ka E. coli me nā hua hope loa (150 mg / L a me 256 mg / L nā moʻomeheu haʻalulu haʻalulu no kāna-NT-GFP a me kāna-NT-PNP, kēlā me kēia), e kūlike me ka mea i hōʻike ʻia. no nā protein ʻē aʻe i hui pū ʻia me NT Ref.30. ʻO kāna-NT-GFP (300mg/mL) a me kāna-NT-PNP (100mg/mL) fusion proteins i hana i nā gels ma hope o 2 hola a me 6.5 hola ma 37 ° C a, ʻo ka mea nui, ʻaʻole i hoʻololi ʻia ka hapa GFP.ʻike ʻia ma hope o ka gelation, me> 70% o ka ikaika fluorescence mua i koe ma hope o ka gelation (Fig. 6a).No ke ana ʻana i ka hana PNP ma kāna-NT-PNP solutions a me nā gels, pono mākou e hoʻoheheʻe i ka protein fusion me NT no ka mea ʻo ka hana enzymatic o ka hoʻomākaukau maʻemaʻe aia ma waho o ka ʻike ʻike o ka assay ma ka gelling concentrations.ʻO ka gel i hanaʻia me kahi hui pū me 0.01 mg / mL His-NT-PNP a me 100 mg / mL NT i mālamaʻia he 65% o ka hana enzymatic mua o nā mea i hoʻopiliʻia (Fig. 6b).Ua paʻa mau ka gel i ka wā o ke ana ʻana (Supplementary Fig. 10).
He ʻano ikaika o ka fluorescence ma mua a ma hope o ka hoʻoheheʻe ʻia ʻana o kāna-NT-GFP (300 mg/mL) a me ka hue i hoʻohuli ʻia i loko o kāna-NT-GFP hydrogel (300 mg/mL) ma lalo o ke kukui ʻike ʻia a me UV.Hōʻike nā kiko i nā ana pākahi (n = 3), hōʻike nā pahu kuhi i ka ʻokoʻa maʻamau.Hōʻike ʻia ka waiwai awelika ma ke kikowaena o nā pahu kuhi.b Ua loaʻa ka hana PNP ma o ka fluorometric analysis me ka hoʻohana ʻana i nā hāʻina a me nā gels i loaʻa iā NT (100 mg/ml) a me kahi hui ʻana i loaʻa ka 0.01 mg/ml kāna-NT-PNP a me 100 mg/ml New Taiwan dollars.Hōʻike ka inset i kahi hue i hoʻohuli ʻia i loko o kahi hydrogel i loko o kāna-NT-PNP (5 mm scale bar).
Maʻaneʻi, hōʻike mākou i ka hoʻokumuʻiaʻana o nā hydrogels mai NT a me nā protein spidroin recombinant'ē aʻe ma o ka hoʻouluʻana i kahi solution protein ma 37 ° C (Figure 1).Hōʻike mākou e pili ana ka gelation me ka hoʻololi ʻana o nā α-helice i loko o nā β-layers a me ka hoʻokumu ʻana i nā fibrils like amyloid (Fig. 3 a me 4).He mea kāhāhā kēia ʻike ʻana ʻo nā NTs he mau puʻupuʻu puni honua ʻelima-helix i ʻike ʻia no ko lākou solubility kiʻekiʻe loa a me ke kūpaʻa kiʻekiʻe ma ka ʻike ʻana> 200 mg/mL ma 4°C no kekahi mau lā27.Eia hou, hoʻololi hou nā NT ma hope o ka denaturation o ka wela ma nā haʻahaʻa protein haʻahaʻa ma µM.E like me kā mākou hopena, pono ka hoʻokumu ʻana o ka fibril i ka hui ʻana o> 10 mg / mL ka nui o ka protein a me ka wela haʻahaʻa kiʻekiʻe (Fig. 1).Ua kūlike kēia me ka manaʻo e hiki ke hoʻokumu ʻia nā fibrils amyloid mai nā polokina i hoʻopili ʻia i loko o kahi ʻāpana wehe ʻia ma muli o nā loli wela ma lalo o nā kūlana physiological 48 .ʻO nā hiʻohiʻona o nā protein i loaʻa i kēia hoʻololi ʻana he insulin49,50, β2-microglobulin, transthyretin a me lysozyme51,52,53.ʻOiai he α-helix ʻo NT i kona mokuʻāina maoli, ma kahi o 65% ​​o ke kaulahao polypeptide i kūpono me ka hoʻokumu ʻana i ka zipper steric (Fig. 4e) 45 .No ka mea, hiki i ka monomer ke hoʻoikaika i ka mobile46, hiki iā ia ke hōʻike i kēia mau ʻāpana amyloidogenic i nā wela haʻahaʻa haʻahaʻa a ma nā kiʻekiʻe kiʻekiʻe o ka protein holoʻokoʻa hiki ke hiki i kahi koʻikoʻi koʻikoʻi no ka hoʻokumu ʻana o ka amyloid fibril54.Ma hope o kēia noʻonoʻo ʻana, ua ʻike mākou i ka pilina maikaʻi ʻole ma waena o ka hoʻonui ʻana o ka spidroin a me ka manawa gelation (Fig. 1c), a inā hoʻopaʻa ʻia ka conformation monomeric NT e nā mutations (NT*, His-NT-L6) a i ʻole ma ka hoʻohui paʻakai, hiki ke pale i ka nā hydrogels hoʻokumu (Fig. 5).
I ka hapanui o nā hihia, nalowale nā ​​fibrils amyloid mai ka hopena ma ke ʻano he precipitate, akā ma lalo o kekahi mau kūlana hiki iā lākou ke hana i nā hydrogels55,56,57.ʻO nā fibrils hoʻoheheʻe hydrogel maʻamau he ʻano kiʻekiʻe a hoʻokumu i nā ʻupena ʻekolu-dimensional paʻa ma o ka molecular entanglement,55,58 e like me kā mākou hopena.No ka hoʻokumu ʻana o ka hydrogel i loko o ka vitro, ʻike pinepine ʻia nā protein a i ʻole he hapa, no ka laʻana, ma ka ʻike ʻana i nā mea hoʻoheheʻe organik, kiʻekiʻe wela (70-90 ° C) a me/a haʻahaʻa pH (1.5-3.0) 59,60,61,62.ʻAʻole koi ʻia nā hydrogels spidroin i hōʻike ʻia ma ʻaneʻi i ka hana koʻikoʻi, ʻaʻole pono lākou i nā mea hoʻohui cross-link e hoʻopaʻa i nā hydrogels.
Ua hōʻike mua ʻia e hana hou ana ka spdroin a me nā QD, ka mea i ʻike ʻia e hoʻololi i ka β-pepa i ka wā o ka wili kilika, e hana i nā hydrogels.Ke hoʻohālikelike ʻia i kā mākou ʻike, ʻoi aku ka lōʻihi o ka manawa incubation a me / a i ʻole ke kiʻekiʻe o ka incubation, a ʻo ka hopena hydrogels he opaque pinepine (Figure 7 and Supplementary Table 1) 37, 38, 63, 64, 65, 66, 67, 68 , 69. Ma waho aʻe o nā manawa gel wikiwiki, ʻoi aku ka maikaʻi o nā hydrogels NT> 300 mg / mL (30%) i nā mea ʻē aʻe a pau i wehewehe ʻia i ka spider silk protein hydrogels, a me nā hydrogels maoli e like me gelatin, alginate (2%), agar (0.5 % ) a me ka collagen.(0.6%) (Figure 7 a me nā Papa Hoʻohui 1 a me 3) 37,39,66,67,68,69,70,71,72,73,74.
Ua hoʻohālikelike ʻia ka manawa gel a me ka modulus elastic o nā hydrogels i kēia haʻawina me nā hydrogels i hoʻokumu ʻia i ka spidroin a me nā hydrogels kūlohelohe i koho ʻia.Hāʻawi ʻia nā kuhikuhi me ka wehewehe ʻana i nā kūlana gelation.APS Ammonium persulfate, lumi wela.ʻIkepili 37, 38, 39, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
ʻIke ʻia ua hoʻomohala nā spiders i nā ala e pale ai i ka spidrin mai ka gelling i ka wā mālama.ʻOiai ke kiʻekiʻe o ka nui o ka protein i loko o ka ʻili siliki, ʻo ka ʻāpana hou nui e pili ana i ke kikowaena kikowaena ʻo ia ka ʻike ʻana o ka NT a me CT i loko o ka ʻeleʻele e pili ana i ka 10-20 mg/ml, ma ka palena o kēia haʻawina.Pono ʻia no ka hoʻokumu ʻana o ka hydrogel in vitro.Eia kekahi, ua hoʻopaʻa ʻia nā ʻano like o nā paʻakai 16 i ka NT, e like me nā kilika silika (Fig. 5b).Ua aʻo ʻia ka conformation NT ma ka cytosol E. coli a ʻike ʻia ʻoi aku ka paʻa o ka pelu ʻia ma mua o ka nānā ʻia ʻana i loko o ka vitro, e hōʻike hou ana i ka paʻakai a i ʻole nā ​​mea ʻē aʻe e pale i kona hōʻuluʻulu ʻana i loko o vivo.Eia nō naʻe, he mea nui ka hiki o nā NT ke hoʻololi i nā fibrils β-sheet no ka hoʻokumu ʻana i nā filament a pono e noiʻi ʻia i nā haʻawina e hiki mai ana.
Ma waho aʻe o nā hiʻohiʻona hou o ka NT-amyloid-like fibril a me ka hoʻokumu ʻana o ka hydrogel i ʻike ʻia i loko o kēia haʻawina, hōʻike pū mākou e loaʻa paha i kēia hanana nā noi biotechnological a me biomedical (Fig. 8).Ma ke ʻano he hōʻoia o ka manaʻo, hoʻohui mākou i ka NT me GFP a i ʻole PNP a hōʻike i ka hana ʻana o ka protein fusion i nā hydrogels ke hoʻomoʻa ʻia ma 37 ° C a ʻo ka GFP a me nā hapa PNP ka nui o kā lākou hana ma hope o ka gelation (Figure 6).ʻO nā Nucleoside phosphorylases nā mea hoʻoheheʻe koʻikoʻi o ka nucleoside analogues75, kahi e kūpono ai kā mākou ʻike no ka ʻoihana biopharmaceutical.ʻO ka manaʻo o ka hōʻike ʻana i nā protein fusion e hana i nā hydrogels māmā ma lalo o nā kūlana maikaʻi e hiki ai i ka hana ʻana o nā hydrogels i hana ʻia me nā waiwai maikaʻi no ka nui o nā noi e like me ka immobilization enzyme, ka hoʻokuʻu ʻana i ka lāʻau lapaʻau a me ka ʻenekini kiko.Eia kekahi, ʻo NT a me NT* nā māka hōʻike maikaʻi30, ʻo ia ka mea hiki ke hoʻohana ʻia ka NT a me kāna mau ʻokoʻa no ka hana kiʻekiʻe-throughput o nā protein fusion soluble a me ka hana ʻana o nā protein target immobilized i 3D hydrogels.
Hiki ke hoʻoheheʻe ʻia ka NT, α-helical a paʻa i nā haʻahaʻa haʻahaʻa (µM) a me 37°C.Ma ka mahana like, akā i ka hoʻonui ʻana i ka nui (> 10 mg / ml), hana ʻo NT i nā gels me nā fibrils like amyloid.Hoʻokumu pū ʻia nā protein fusion NT i nā gel fibrillar me nā ʻāpana fusion holoʻokoʻa, e ʻae ana i nā protein like ʻole e hoʻoneʻe ʻia i nā hydrogels 3D me ka hoʻohana ʻana iā NT.Ma lalo: NT (PDB: 4FBS) a me nā kiʻi kiʻi o nā ʻupena fiber a me nā ʻōnaehana protein e pili ana (manaʻo ʻia a ʻaʻole huki ʻia i ka unahi, GFP PDB: 2B3Q, 10.2210/pdb2B3Q/pdb; PNP PDB: 4RJ2, 10.2210/pdb4RJ2/pdb).
ʻO nā mea i kūkulu ʻia (e nānā i ka Papa Hoʻohui 4 no kahi papa inoa piha me nā kaʻina amino acid) ua cloned i plasmid pT7 a hoʻololi ʻia i E. coli BL21 (DE3).E. coli i loaʻa nā plasmids engineered ua inoculated i loko o Luria broth hoʻohui me kanamycin (70 mg / l) a ulu i ka pō ma 30 ° C a me 250 rpm.Hoʻokomo ʻia ka moʻomeheu i ka 1/100 i loko o ka medium LB i loaʻa ka kanamycin a hoʻoulu ʻia ma 30 ° C a me 110 rpm a hiki i ka OD600 i 0.8.No nā haʻawina NMR, ua ulu ʻia ka bacteria ma M9 liʻiliʻi liʻiliʻi me 2 g D-glucose 13C (Aldrich) a me 1 g o ka ammonium chloride 15N (Cambridge Isotope Laboratories, Inc.) no ka hoʻopaʻa inoa ʻana me nā isotopes.E hoʻohaʻahaʻa i ka mahana a hiki i 20 degere Celsius a hoʻoulu i ka hōʻike protein me 0.15 mM isopropylthiogalactopyranoside (ka hopena hope).Ma hope o ka hōʻike protein i ka pō, ua ʻohi ʻia nā cell ma 7278 × g, 4 ° C no 20 min.Ua hoʻokuʻu hou ʻia nā pellet cell ma 20 mM Tris-HCl, pH 8, a me ka hau a hiki i ka hoʻohana hou ʻana.Hoʻopili ʻia nā pūnaewele i hoʻoheheʻe ʻia me ka hoʻohana ʻana i kahi mea hoʻonāwaliwali (TS series machines, Constant Systems Limited, ʻEnelani) ma 30 kPa.A laila ua centrifuged nā lysates ma 25,000 g no 30 mau minuke ma 4 ° C.No ka NTMiSp, ua hoʻokuʻu ʻia ka pellet ma 2 M urea, 20 mM Tris-HCl, pH 8, a sonicated no 2 min (2 s on / off, 65%), a laila centrifuged hou i 25,000 xg, 4 ° C. i loko. 30 min.Hoʻokomo ʻia ka supernatant ma luna o kahi kolamu Ni-NTA, holoi ʻia me 20 mM Tris-HCl, 2 mM imidazole, pH 8, a hope loa ua hoʻoheheʻe ʻia ka protein me 20 mM Tris-HCl, 200 mM imidazole, pH 8. No ka hana ʻana i ka NT2RepCT a NTCT, thrombin digestion e hoʻolauna i ka pūnaewele (ThrCleav) ma waena o kāna a me NT.Loaʻa nā pae ʻāpana Thrombin i kāna-NT-ThrCleav-2Rep (hana 2Rep), kāna-thioredoxin-ThrCleav-NT (hana NT), kāna-thioredoxin-ThrCleav-CT (hana CT), kāna-Thioredoxin-ThrCleav-NT .* (hana NT*), His-Thioredoxin-ThrCleav-NTA72R (hua NTA72R), His-Thioredoxin-ThrCleav-NTFlSp (hua NTF1Sp), a me His-Sulphur Redoxin-ThrCleav-NTMiSp (hua NTMiSp).Hoʻopili ʻia nā mea kūkulu me ka thrombin (1: 1000) a hoʻopaʻa ʻia i ka pō ma 4 ° C. me 20 mM Tris-HCl, pH 8, me ka hoʻohana ʻana i kahi membrane Spectra / Por dialysis me kahi paepae kaumaha mole o 6-8 kDa.Ma hope o ka dialysis, hoʻouka ʻia ka hopena ma luna o kahi kolamu Ni-NTA a ʻohi ʻia ka effluent i loaʻa ka protein o ka hoihoi.Ua hoʻoholo ʻia ka nui o ka protein ma ke ana ʻana i ka absorbance UV ma 280 nm me ka hoʻohana ʻana i ka coefficient extinction o kēlā me kēia protein, koe wale nō ka NTF1Sp, ka mea i hoʻohana i ka hōʻike Bradford e like me ke kaʻina hana.Hoʻoholo ʻia ka maʻemaʻe e SDS polyacrylamide (4-20%) gel electrophoresis a me Coomassie brilliant blue staining.Hoʻopili ʻia nā protein me ka hoʻohana ʻana i nā kānana centrifuge (VivaSpin 20, GE Healthcare) ma 4000 xg me kahi ʻoki paona 10 kDa molecular i loko o 20 mau minuke.
E hoʻoheheʻe i ka solution protein a hoʻopaʻa pono i 150 µl i loko o kahi hue septum maʻemaʻe 1 ml (8 x 40 mm Thermo Scientific).Hoʻopaʻa ʻia nā paipu a hoʻopaʻa ʻia me ka parafilm i mea e pale ai i ka mahuʻi.Hoʻokomo ʻia nā laʻana (n = 3) ma 37 ° C a i ʻole 60 ° C a hoʻohuli ʻia i kēlā me kēia manawa e nānā i ka gelation.Hoʻokomo ʻia nā laʻana ʻaʻole gel i hoʻokahi pule.E hoemi i na paa disulfide NTMiSp me 10 mM DTT no 10 µM protein.No ka hoʻopili ʻana i ka gelation o nā uhi silika spider maoli, ua ʻoki ʻia ka spider bridge spider, ua hoʻokomo ʻia nā glands ampullated nui ʻelua i 200 μl o 20 mM Tris-HCl buffer pH 8 a ʻoki ʻia e ʻae i ka uhi e hoʻokaʻawale mai nā glands..Hoʻoheheʻe ʻia nā mea i loko o ke kīpē i loko o ka buffer, 50 µl no ka hoʻoholo ʻana i ke kaumaha maloʻo (ma ka hoʻoulu ʻana o nā hue hāmama ma 60 °C i ke kaumaha mau) a me 150 µl no ka gelation ma 37 °C.
Hana ʻia ka geometry / mea hana me ke kila kila me ka hoʻohana ʻana i kahi pā like me ke anawaena o luna o 20 mm a me kahi āpau o 0.5 mm.E wela i ka hāpana mai 25 °C a i 45 °C a hoʻi i 25 °C ma ke ana o 1 °C i kēlā me kēia minuke me ka hoʻohana ʻana i ka pā Peltier lalo kila kila.Ua lawe ʻia nā ana vibrational ma ke alapine o 0.1 Hz a ma ka ʻāpana viscoelastic linear o ka mea ma kahi kānana o 5% a me 0.5% no nā laʻana o 100 mg / mL a me 300-500 mg / mL.E hoʻohana i kahi keʻena haʻahaʻa maʻamau e pale i ka hoʻoheheʻe ʻana.Ua kālailai ʻia ka ʻikepili me ka Prism 9.
No ka hōʻiliʻili ʻana i nā spectra infrared (IR) ma ka lumi wela mai 800 a 3900 cm–1.Hoʻomaʻemaʻe ʻia ka mea ATR, a me ke ala māmā ma o ka spectrometer me ka ea kānana maloʻo ma mua a i ka wā o ka hoʻokolohua.ʻO nā hāʻina (500 mg / mL e hōʻemi i nā peaks absorption wai i ka spectra) ua paipu ʻia ma luna o nā kristal, a ua hoʻokumu ʻia nā gels (500 mg / mL) ma mua o ke ana ʻana a laila hoʻoili ʻia i nā kristal (n = 3).Ua hoʻopaʻa ʻia nā scans 1000 me ka hoʻonā ʻana o 2 cm-1 a me ka pōʻai hana ʻole o 2. Ua helu ʻia ka derivative lua me ka hoʻohana ʻana i ka OPUS (Bruker) me ka hoʻohana ʻana i kahi laulima o ʻeiwa mau helu.Ua maʻamau ka spectra i ka māhele hoʻohui like ma waena o 1720 a me 1580 cm-1 me ka hoʻohana ʻana iā F. Menges "Spectragryph - Optical Spectroscopy Software".I ka ATR-IR spectroscopy, pili ka hohonu o ke komo ʻana o kahi kaola infrared i loko o kahi laʻana i ka helu hawewe, a ʻoi aku ka ikaika o ka lawe ʻana ma nā helu haʻahaʻa haʻahaʻa ma mua o nā helu hawewe kiʻekiʻe.ʻAʻole i hoʻoponopono ʻia kēia mau hopena no ka spectra i hōʻike ʻia ma Fig.3 no ka mea, liʻiliʻi loa lākou (Fig 4).Ua helu ʻia ka spectra i hoʻoponopono ʻia no kēia kiʻi me ka polokalamu Bruker OPUS.
Ma ke kumu, hiki ke helu piha i ka conformations protein ma hope o ka deconvolution hilinaʻi o nā mea i loko o ka amide I peak.Eia naʻe, ke kū nei kekahi mau pilikia ma ka hana.Hiki ke ʻike ʻia ka walaʻau ma ke kiko kikoʻī ma ke ʻano he mau piko (hewa) i ka wā deconvolution.Eia kekahi, ʻo ke kiʻekiʻe ma muli o ka piʻi ʻana o ka wai e like me ke kūlana o ka amide I peak a loaʻa paha ka nui like no nā laʻana i loaʻa ka nui o ka wai, e like me ka aqueous gel i aʻo ʻia ma aneʻi.No laila, ʻaʻole mākou i hoʻāʻo e hoʻopau loa i ka amide I peak, a pono e noʻonoʻo ʻia kā mākou nānā ʻana i ke kākoʻo ʻana i nā ʻano hana ʻē aʻe e like me ka NMR spectroscopy.
Hoʻopili ʻia nā hopena o 50 mg/ml NT a me kāna-NT2RepCT i ka pō ma 37 ° C.A laila hoʻoheheʻe ʻia ka hydrogel me 20 mM Tris-HCl (pH 8) i kahi ʻano o 12.5 mg / ml, hoʻoluliluli maikaʻi ʻia a paipu ʻia e wāwahi i ka gel.A laila, ua hoʻoheheʻe ʻia ka hydrogel i 10 mau manawa me 20 mM Tris-HCl (pH 8), 5 μl o ka hāpana i hoʻopili ʻia i kahi pahu keleawe i uhi ʻia me formvar, a ua hoʻoneʻe ʻia ka hāpana keu me ka pepa blotting.Ua holoi ʻia nā laʻana me 5 µl o ka wai MilliQ a hoʻopaʻa ʻia me 1% uranyl formate no 5 mau minuke.Wehe i ka ʻeleʻele me ka pepa absorbent, a laila hoʻomaloʻo i ka ʻea.Ua hana ʻia ke kiʻi kiʻi ma kēia mau pahu me ka FEI Tecnai 12 Spirit BioTWIN e hana ana ma 100 kV.Ua hoʻopaʻa ʻia nā kiʻi ma x 26,500 a me x 43,000 hoʻonui me ka Veleta 2k × 2k CCD kamera (Olympus Soft Imaging Solutions, GmbH, Münster, Kelemānia).No kēlā me kēia hāpana (n = 1), ua hoʻopaʻa ʻia nā kiʻi 10-15.Ua hoʻohana ʻia ʻo ImageJ (https://imagej.nih.gov/) no ka nānā ʻana i nā kiʻi a me ke ana ʻana o nā anawaena fiber (n = 100, nā ʻokoʻa ʻokoʻa).Ua hoʻohana ʻia ʻo Prism 9 e hana i nā hoʻāʻo-t ʻole (ʻelua-huelo).ʻO ka mean His-NT2RepCT a me NT fibrils he 11.43 (SD 2.035) a me 7.67 (SD 1.389) nm.ʻO ka wā hilinaʻi (95%) ʻo -4.246 a -3.275.degere o ke kuokoa = 198, p <0.0001.
Ua ana ʻia he 80 µl o nā laʻana wai i loaʻa he 10 µM thioflavin T (ThT) ma ka ʻekolu (n = 3) ma lalo o nā kūlana paʻa me ka hoʻohana ʻana i nā papa lalo ʻeleʻele Corning 96-well ʻeleʻele (Corning Glass 3881, USA).Ua hoʻopaʻa ʻia nā ʻokoʻa fluorescence me ka hoʻohana ʻana i kahi kānana excitation 440 nm a me kahi kānana emission 480 nm (FLUOStar Galaxy mai BMG Labtech, Offenburg, Kelemānia).ʻAʻole i hoʻopiha ʻia ka hōʻailona ThT, no ka mea, ua hana ʻia nā hoʻokolohua me nā ʻano ʻokoʻa like ʻole o ka ThT me ka hoʻololi ʻole i ka ikaika o ka hōʻailona.E hoʻopaʻa i ka hoʻopaʻa ʻana ma 360 nm no ke ana ʻohu.No nā hoʻokolohua seeding, ua hoʻokumu ʻia nā gels 100 mg / mL ma 37 ° C., hoʻomaha hou ʻia, a hoʻohana ʻia no ka lūlū ʻana ma nā ratio molar o 5%, 10%, a me 20%.Ua kālailai ʻia ka ʻikepili me ka Prism 9.
E hoʻoheheʻe i nā waihona o His-NT2RepCT a me NT >100 mg/mL ma ka hau a kānana ma o kahi kānana 0.22 µm.Ua helu ʻia nā concentration ma ke ana ʻana i ka absorbance ma 280 nm me ka hoʻohana ʻana iā Nanodrop.I loko o nā pūnāwai o ka 96-well black non-binding plate (Corning) me kahi māmā o lalo, ua hoʻoheheʻe ʻia nā laʻana i 20 mg/ml ma 20 mM Tris-HCl pH 8 a hui pū ʻia me 5 μM ThT (ka hopena hope), ka nui o ka laʻana. 50 μl ka nui.Ua kiʻi ʻia nā laʻana i kēlā me kēia 10 mau minuke ma 37 °C ma kahi microscope CellObserver (Zeiss) me ke kahawai māmā i hoʻouna ʻia a me ka FITC excitation a me nā hoʻonohonoho kānana hoʻokuʻu no ke kiʻi ThT.Hoʻohana ʻia kahi lens 20x/0.4 no ke kiʻi ʻana.Ua hoʻohana ʻia ʻo Zen Blue (Zeiss) a me ImageJ (https://imagej.nih.gov/) no ka nānā ʻana i nā kiʻi.Ua hoʻomākaukau pū ʻia nā gels mai NT a me His-NT2RepCT solutions ma kahi ʻano o 50 mg / mL i loaʻa 20 mM Tris pH 8 a me 5 µM ThT a incubated ma 37 ° C no 90 min.Ua hoʻoneʻe ʻia nā ʻāpana gel i kahi pūnāwai hou e loaʻa ana he 20 mM Tris, pH 8, a me 5 μM ThT i loko o kahi paʻa ʻeleʻele ʻeleʻele 96 maikaʻi maikaʻi loa.E kiʻi i nā kiʻi ʻōmaʻomaʻo ʻōmaʻomaʻo a me nā kiʻi māla ma ka 20x/0.4 kiʻekiʻe.Ua hoʻohana ʻia ʻo ImageJ no ka nānā ʻana i nā kiʻi.
Loaʻa ka NMR spectra i ka 310 K ma kahi 600 MHz Bruker Avance Neo spectrometer i lako me kahi QCI Quadrupole Resonance Pulsed Gradient Field Cryoprobe (HFCN).NMR laʻana i loaʻa 10 mg/mL homogeneous protein i kapa ʻia me 13C, 15N, hoʻoheheʻe ʻia i 20 mM Tris-HCl (pH 8), 0.02% (w/v) NaN3, 5% DO (v/v), (n = 1) .Ua hoʻohana ʻia nā hoʻololi kemika o NT2RepCT ma ka pH 6.7 no ke kau ʻana i ka piko 23 i ka spectrum 2D o 15N-HSQC.
Ua hoʻopaʻa ʻia nā ʻano hydrogels i kapa ʻia he 13C, 15N-labeled ma ka Bruker Avance III HD spectrometer ma 800 MHz me ka 3.2 mm 13C/15N{1H} electronless probe.Ua hoʻomaluʻia ka mahana laʻana me ka hoʻohanaʻana i kahi kahe kinoea wela ma 277 K. Loaʻaʻia nāʻano dipole rotational resonance (DARR) 76 a me ka hoʻohui houʻana o ka lekiō (RFDR) 77 ma nā alapine MAS o 12.5 kHz a me 20 kHz.Hoʻohana ʻia ka polarization keʻa (CP) mai 1H a 13C me ka laina laina mai 60.0 a 48.0 kHz ma 1H, 61.3/71.6 kHz ma 13C (ma 12.5/20 kHz MAS) a me ka manawa pili 0.5-1 ms.Ua hoʻohana ʻia ʻo Spinal6478 decoupling ma 73.5 kHz i ka wā o ka hōʻiliʻili ʻikepili.ʻO 10 milliseconds ka manawa i loaʻa ai a he 2.5 kekona ka lōʻihi o ka pōʻai.Ua hoʻonohonoho ʻia nā pilina Cα/Cβ pili hoʻokahi i ʻike ʻia ma ka spectra RFDR ma muli o ke ʻano o ke koena-type hoʻololi kemika a me ka hoʻopili hoʻohui ʻia ma ka ʻano DARR spectra.
Ua hoʻohana ʻia ka waihona Zipper79 (https://services.mbi.ucla.edu/zipperdb/) no ka loiloi ʻana i nā ʻano flutter a me ka ikaika Rosetta no NT, NTFlSp, a me NTMiSp.Hoʻopili ka waihona Zipper i ka Rosetta Energy80, ka mea e hoʻohui i kekahi mau hana ikehu manuahi e hoʻohālike a nānā i ka ʻōnaehana protein.ʻO ka pae ikehu o -23 kcal/mol a i ʻole ka haʻahaʻa e hōʻike ana i kahi kiʻekiʻe o ka fibrillate.ʻO ka ikehu haʻahaʻa, ʻoi aku ka paʻa o nā kaula β ʻelua i ka conformation zipper.Eia kekahi, ua hoʻohana ʻia ka Waltz algorithm e wānana i nā ʻāpana amyloidogenic ma NT, NTFlSp a me NTMiSp Ref.81. (https://waltz.switchlab.org/).
Ua hui pū ʻia ka ʻea protein NT me 2-(N-morpholino)ethanesulfonic acid (MES) buffer ma ka pH 5.5 a me 6.0 e hoʻohaʻahaʻa i ka pH i ka pH 6 a me 7.ʻO ka hoʻopaʻa ʻana o ka protein hope loa he 100 mg/ml.
Hana ʻia nā ana ma kahi J-1500 CD spectrometer (JASCO, USA) me ka hoʻohana ʻana i kahi cuvette 300 μL me kahi ala optical o 0.1 cm.Ua hoʻoheheʻe ʻia nā protein i 10 μM (n = 1) ma 20 mM phosphate buffer (pH 8).No ka hoʻopaʻa ʻana i ka paʻa o ka protein i mua o ka paʻakai, ua ʻike ʻia nā proteins ma ke ʻano like (n = 1) ma 20 mM phosphate buffer (pH 8) i loaʻa iā 154 mM NaF a i ʻole NaCl, kēlā me kēia.Ua hoʻopaʻa ʻia nā kiʻi wela ma 222 nm mai 25 ° C a i 95 ° C me ka wikiwiki hoʻomehana o 1 ° C / min.Ua helu ʻia ka ʻāpana o nā protein i hoʻopili ʻia me ka hoʻohana ʻana i ke ʻano (KDmeasure – KDfinal)/(KDstart – KDfinal).Eia kekahi, ʻelima spectra i hoʻopaʻa ʻia no kēlā me kēia hāpana mai 260 nm a 190 nm ma 25 ° C a ma hope o ka hoʻomehana ʻana i 95 ° C.Elima spectra i awelika, smoothed a hoohuli i ka molar ellipticity.Ua kālailai ʻia ka ʻikepili me ka Prism 9.
Ua ana ʻia ka ikaika o ka fluorescence o kāna-NT-GFP (300 mg/mL, 80 µL) ma ka ʻekolu (n = 3) ma nā pā 96-well Corning me kahi lalo ʻeleʻele ʻeleʻele (Corning Glass 3881, USA) ma lalo o nā kūlana static.E ana i nā laʻana me kahi mea heluhelu pā e pili ana i ka fluorescence me ka lōʻihi hawewe o 395 nm a hoʻopaʻa i ka hoʻokuʻu ʻana ma 509 nm ma mua o ka gelation a me 2 mau hola ma hope ma 37°C.Ua kālailai ʻia ka ʻikepili me Prism 9.
Ua hoʻohana ʻia ka purine nucleoside phosphorylase activity assay kit (fluorometric method, Sigma Aldrich) e like me nā ʻōlelo a ka mea hana.No ke ana ʻana i ka hana i loko o nā gels a me nā haʻina e loaʻa ana i kāna-NT-PNP, e hui pū i ka 10 ng o His-NT-PNP me 100 mg/mL NT i ka nui o 2 µL no ka mea ua hāʻawi ka gel i kahi hōʻailona ma luna o ka wā ʻike o ka set.Hoʻokomo ʻia nā mana no nā gels a me nā hoʻonā me ka ʻole o kāna-NT-PNP.Ua hana ʻia nā ana ʻelua (n = 2).Ma hope o ke ana ʻia ʻana o ka hana, ua wehe ʻia ka hui ʻana a paʻi ʻia ke kiʻi e hōʻoia i ka paʻa ʻana o ka gel i ka wā o ke ana.Ua kālailai ʻia ka ʻikepili me ka Prism 9.
No ka ʻike hou aku e pili ana i ka hoʻolālā haʻawina, e ʻike i ka abstract study Nature i hoʻopili ʻia i kēia ʻatikala.
Hōʻike nā kiʻi 1 a me 2 i ka ʻikepili mua.1c, 2a–c, 3a, b, e–g, 4, 5b, d, f, a me 6, Hoʻohui Fig.3, fig.5a, d, fig hou.6 a me ka fig hou.8. Hoʻokipa ʻia ka ʻikepili ʻikepili mai kēia haʻawina ma ka waihona Zenodo https://doi.org/10.5281/zenodo.6683653.Ua kau ʻia nā ʻikepili NMR i loaʻa ma kēia haʻawina i ka waihona BMRGig ma lalo o ka ID komo bmrbig36.Ua lawe ʻia nā hale o GFP a me PNP mai PDB (GFP 2B3Q, PNP 4RJ2).
Rising, A. a me Johansson, J. Ke wili ana i ke kilika spider artificial.Kemika Lahui.biology.11, 309–315 (2015).
Babb, PL et al.Hōʻike ka Nephila clavipes genome i ka ʻokoʻa o nā genes silika spider a me ko lākou ʻano paʻakikī.Genette Lahui.49, 895–903 (2017).