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Protein silk udide recombinant (protein silk ududo) nwere ọtụtụ ngwa nwere ike ime na mmepe nke biomaterials ọhụrụ, mana ọdịdị multimodal na mkpokọta na-adịkarị mfe na-eme ka ha sie ike inweta ma dị mfe iji.N'ebe a, anyị na-akọ na recombinant miniature spidroin proteins na, ihe dị mkpa, N-terminal ngalaba (NT) n'onwe ya na-etolite ngwa ngwa na-akwado onwe ya na transperent hydrogels na 37 Celsius.protein fusion nke nwere NT na protein fluorescent green ma ọ bụ purine nucleoside phosphorylase na-etolite protein fusion zuru oke.Hydrogels.Nsonaazụ anyị na-egosi na recombinant NT na protein fusion na-enye mkpụrụ okwu dị elu ma na-enye hydrogels nwere njirimara mara mma dị ka nghọta, gelation na-enweghị crosslinking, na mgbochi ozugbo nke protein na-arụ ọrụ na njupụta dị elu.
Ududo nwere ihe dị ka ụdị silk asaa dị iche iche, nke ọ bụla na-emepụta ụdị silk dị iche iche.Ụdị silk asaa niile nwere protein silk ududo (spidroins) ihe dị ka 6000 residues ogologo ma nwee nnukwu mpaghara ikwugharị nke etiti gbara gburugburu N- na C-terminal ngalaba (NT na CT) 1,2.Ụdị silk nke a na-amụkarị, bụ ampulla nke mbụ, bụ nke bụ isi ampulla gland na-emepụta.N'ime gland a, monolayer nke mkpụrụ ndụ epithelial na-emepụta proteins spidroin ma zoo ha n'ime lumen nke gland, ebe ha nọ n'ụdị soluble (doping) na oke elu (30-50% w / v) 3,4.A na-arụrịta ụka na nhazi na nhazi nke protein spidroin bụ isi nke ampullar dị na gland, ma ọtụtụ ihe akaebe na-egosi na ọ dị n'ozuzu helical na / ma ọ bụ ihe na-eme ka ọkpụkpụ na-egbuke egbuke na micellar ma ọ bụ lamellar 5,6,7,8,9,10.Ọ bụ ezie na ngalaba ugboro ugboro na-ahazi ihe eji arụ ọrụ nke eriri silk, na-akpụ β-sheet nanocrystals na amorphous structures11,12,13,14,15, ngalaba njedebe na-achịkwa eriri silk na nzaghachi na ọnọdụ mgbanwe n'akụkụ silk gland16,17,18.Site n'ịchịkwa nhazi silk, 19. A na-echekwa ngalaba njedebe na evolushọn na ọrụ ha nwere ike ịdị na-ahụkarị na protein spidroin niile 2,20,21.Mgbe ị na-agafe na gland, pH nke spidroin na-ebelata site na ihe dịka 7.6 ruo <5.716 ma na-abawanye site na ịkwa akwa ma gbatịa site na mmegharị site na eriri nke na-eji nwayọọ nwayọọ na-ebelata.Na ngwọta, CT bụ α-helical constitutive parallel dimer17, ma na nzaghachi na obere pH na shear agha, CT na-ekpughere ma gbanwee β-layers16, 17, ikekwe na-akpalite β-layers na mpaghara ugboro ugboro nke Ntụgharị 16. NT bụ monomeric n'okpuru. ọnọdụ na-egosipụta ọnọdụ na lumen nke gland na mediate solubility nke spidroin, ma na mbelata pH, protonation nke a ọnụ ọgụgụ nke carboxylic acid n'akụkụ agbụ na-eduga na dimerization nke NT na a pKa nke mkpokọta 6.5, si otú stabilize NT na idozi spidroin na nnukwu. ọnụọgụgụ.netwọk 16,18.Ya mere, NT na-arụ ọrụ dị mkpa na nhazi nke filament, na-agbanwe site na monomer na mkpuchi na dimer na fiber23,24,25.NT na-anọgide na-enwe ike soluble na helical n'okpuru ọnọdụ niile a mụrụ ruo taa16, 18, 19, 20, 26, 27, 28, 29, bụ nke kpaliri mmepe ya dị ka akara na-akwalite solubility maka mmepụta nke proteins heterologous.
Recombinant Obere udide silk protein, nke nwere otu NT, otu obere ikwugharị mpaghara, otu CT, na mkpado His6 (His-NT2RepCT) maka ịdị ọcha, dị ka soluble na aqueous nchekwa dị ka nwa afọ ududo silk protein na-eṅomi nwa afọ dị mkpa àgwà nke silk udide. .mkpuchi 25.31.Enwere ike ịgbanye ya-NT2RepCT n'ime eriri na-aga n'ihu site na iji igwe biomimetic nke a na-etinye mkpuchi pH 8 soluble n'ime pH 525,32,33,34,35 mmiri ịsa ahụ.Bioreactor fermentation nke E. coli na-ekwupụta Ya-NT2RepCT na usoro ọgwụgwọ ga-esochi rụpụtara> 14 g/L mkpụrụ mgbe emechara ya ọcha.A na-ekwu na ọ bụ NT23, 25, 34, mkpụrụ dị elu, solubility dị elu na nzaghachi zuru oke nke His-NT2RepCT na ọnọdụ acidic.
N'ebe a, anyị na-akọ nhazi ngwa ngwa nke hydrogels transperent sitere na protein spidroin recombinant, gụnyere NT naanị, site n'itinye ngwọta protein na 37 Celsius.N'iji thioflavin T fluorescence (ThT), Fourier na-agbanwe infrared spectroscopy (FTIR), nuklia magnetik resonance spectroscopy (NMR) na nnyefe electron microscopy (TEM), anyị chọpụtara na NT na microspider proteins na-enweta mgbanwe nhazi n'ime β-ibé akwụkwọ na amyloid dị ka fibrils. mgbe a na-emepụta gels.Na mgbakwunye, protein fusion nke NT na protein fluorescent green (GFP) ma ọ bụ purine nucleoside phosphorylase (PNP) na-etolite hydrogels nwere iberibe ngwakọta na-arụ ọrụ zuru oke.Ngosipụta dị elu na ndị agha heterologous, yana nhazi ngwa ngwa nke hydrogels n'okpuru ọnọdụ physiological, na-emepe ohere nke mmepụta hydrogels dị ọnụ ala na-arụ ọrụ nhazi.
N'adịghị ka ọtụtụ protein recombinant spidroin36, His-NT2RepCT kwụsiri ike na ihe nchekwa Tris-HCl na pH 8 ma nwee ike itinye uche ruo 500 mg/mL na-enweghị ọdịda25.Ya mere, ọ tụrụ anyị n'anya ịhụ na protein a na-etolite ngwa ngwa n'ụzọ doro anya, hydrogels na-akwado onwe ya mgbe a na-etinye ya na 37 ° C (Fig 1b-d).Nnyocha ndị ọzọ gosiri na gelation His-NT2RepCT mere n'ọtụtụ protein protein (10-300 mg / mL) nakwa na ntinye uche a na-ejikọta ya na oge gelation (Fig 1c na Fig. 1).Iji chọpụta akụkụ nke His-NT2RepCT mediate hydrogel formation, anyị wee nyochaa ngalaba nke ọ bụla n'otu n'otu na n'ụdị dị iche iche site na iji flask inversion assay (Figure 1a,b).Akụkụ niile a nwalere nke spidroin recombinant kpụrụ gels (na ntinye protein nke 300 mg / mL) n'ime ihe na-erughị 1 h, ma e wezụga maka 2Rep (Fig. 1b).Nke a na-egosi na NT na CT naanị, na nchikota, ma ọ bụ jikọtara ya na ikwughachi, nwere ike gel na 37 Celsius C na mkpado His6 adịghị emetụta usoro a ruo n'ókè ọ bụla.N'iburu echiche a na-ahụkarị na NT bụ protein na-agbaze nke ukwuu ma kwụsie ike, yana akụkọ ndị gara aga nke spidroin hydrogels recombinant ekwupụtala mmetụta gelation na mgbanwe mgbanwe na mpaghara ugboro ugboro na / ma ọ bụ CT, NT n'onwe ya nwere ike.Nchọpụta nke gelation bụ ihe a na-atụghị anya ya.Tebụl mgbakwunye 1) 37, 38, 39. N'ụzọ dị ịrịba ama, NT abanyelarị n'ime nkeji iri na ntinye nke ≥ 300 mg/mL (Fig 1c).Nnwale inversion nke vial na ntinye dị iche iche nke NT gosiri na na> 50 mg / mL ngwọta NT na-agba ọsọ ọsọ karịa His-NT2RepCT na ntinye kwekọrọ (w / v, Figure 1c).
Ihe nlere anya nke spidroin dị iche iche a mụrụ n'ọrụ a.b Gel oge na 37 Celsius C maka protein spidroin dị iche iche recombinant (300 mg/mL) kwadoro site na ịtụgharị vial.CT gel ozugbo na-enweghị incubation (<300 mg / ml), 2Rep precipitates (300 mg / ml, 5 mm scale).c Gel oge nke His-NT2RepCT na NT na protein egosipụtara na 37 Celsius.d Foto nke His-NT2RepCT na NT hydrogels nwere spider na leta "NT" nke e bipụtara n'okpuru, n'otu n'otu (ma 200 mg/mL, ọnụ ọgụgụ mmanya 5 mm).
Hydrogels kpụrụ site na protein spidroin dị iche iche recombinant nwere agba dịtụ iche, na nlele anya gba ọtọ na-egosi ogo nghọta dị iche iche (Fig 1b).Gel NT doro anya nke ọma ebe gel ndị ọzọ na-aghọ opaque.Ya-NT2RepCT na NT gels tụbara n'ime cylindrical tubes nwere ike wepụ na ebu na-emebibeghị (Fig. 1d).
Iji nwalee ma ihe mkpuchi silk ududo eke n'okpuru ọnọdụ ndị achọpụtara ugbu a na-akpata gelation nke protein spidroin recombinant, a na-anakọta mkpuchi site na nnukwu ampulla gland nke ududo akwa Sweden (Larinioides sclopetarius).A na-echekwa mkpuchi mkpuchi na 20 mM Tris-HCl buffer na 50 mg / mL (dabere na nha akọrọ), ma ọ dịghị gelation a hụrụ n'oge 21 ụbọchị incubation na 37 Celsius C (Ngbakwunye 2a).
Iji chọpụta gels ndị a, enwere ike iji nha rheological iji mụọ usoro gelation ma chọpụta ihe niile eji arụ ọrụ.Karịsịa, nyochaa modulus nchekwa (elasticity) na okpomọkụ dị elu nwere ike inye ozi gbasara okpomọkụ gelling yana njirimara viscoelastic nke mkpuchi.Okpomọkụ ịrị elu nnwale (iji 1 ° C / min na 25-45 ° C, dabere na gara aga ọmụmụ iji eke silk ngwaahịa ngwọta) 40,41 gosiri na nchekwa moduli nke His-NT2RepCT na NT ngwọta mụbara na-amụba okpomọkụ.a mụbara (Fig. 2 na mgbakwunye Fig. 3).N'ụzọ doro anya, modul NT malitere itolite na okpomọkụ dị ala ma e jiri ya tụnyere His-NT2RepCT, kwekọrọ na oge gel ngwa ngwa a hụrụ mgbe NT jikọtara ya na His-NT2RepCT ozugbo na 37 ° C (Njirimara 1).Mgbe ọdịda okpomọkụ na-esote, modul nchekwa ahụ alaghachighị n'ụkpụrụ dị ala ma nọrọ n'elu modul ọnwụ (lee Mgbakwunye Fig. 3), na-egosi gelation kwụsiri ike na-enweghị ike ịgbanwe.Mgbe gelation gasịrị, modul ikpeazụ nke na-agbanwe agbanwe sitere na 15 ruo 330 kPa maka His-NT2RepCT hydrogels na ntinye nke 100-500 mg / ml, na njedebe ikpeazụ maka NT hydrogels (100-500 mg / ml) sitere na 2 ruo 1400. kPa (Fig. , 2 na data ramp zuru ezu) lee Mgbakwunye 3).
Mgbanwe na okpomọkụ n'oge nha His-NT2RepCT (300 mg/mL) na b NT (300 mg/mL) na ịma jijiji.Àkú ndị ahụ na-egosi ọnọdụ okpomọkụ, na ncha ọkụ nke data modul nchekwa na-egosi nnwale na ọnụ ọgụgụ dị ala maka ngwá ọrụ karịa nke onye nrụpụta kwuru kpọmkwem, nke bụ ihe kpatara ụzụ na-abawanye.c Mkpokọta njedebe-modul nke His-NT2RepCT na NT mgbe okpomọkụ dị elu (100, 300, na 500 mg/mL).A na-agụ ọgụgụ modul niile na ugboro 0.1 Hz.
Dị ka ụzọ nwere ike isi nyochaa mgbanwe mgbanwe na-ejikọta na gelation, anyị dekọrọ FTIR spectra nke His-NT2RepCT na NT tupu na mgbe gelation gasịrị na 37 ° C (Figure 3a,b).Dị ka a na-atụ anya ya, ụdịdị nke His-NT2RepCT na NT ngwọta kwekọrọ na protein ndị na-egosi α-helix/random coil Ọdịdị nke abụọ, yana eriri akpọpụtara na 1645 cm-1.Maka hydrogels abụọ, gelation mere ka e guzobe ogwe aka abụọ n'etiti m band na ihe dịka 1617 cm-1 na 1695 cm-1 (Fig 3a, b), na-egosi nhazi nke mgbochi β-ibé akwụkwọ.A pụkwara ịhụ mgbanwe ndị a n'ụzọ doro anya na ụdị nke abụọ nke abụọ na ọdịiche gelation spectra (Mgbakwunye 4b).Asụsụ abụọ nke NT β-layer pụtara karịa nke His-NT2RepCT, na-egosi na mkpokọta ọdịnaya nke β-layer bands na NT hydrogel dị elu karịa nke NT2RepCT hydrogel.
a FTIR absorption ụdịdị nke His-NT2RepCT na b NT (ma 500 mg/mL) tupu (ngwọta) na mgbe (gel) incubation na 37°C.c TEM onyonyo eweghachitere 50 mg/ml NT2RepCT gels na d NT.Ogwe ọnụ ọgụgụ 200 nm.e Fiber diameters nke His-NT2RepCT na NT hydrogels.n = 100 fibrils tụrụ atụ, p <0.0001.Ogwe mperi na-egosi ndapụta ọkọlọtọ.Ebe etiti ogwe njehie bụ ihe pụtara.A na-eji t-ule na-ejikọtaghị ọnụ (ọdụdụ abụọ) maka nyocha ọnụ ọgụgụ.f ThT fluorescence nke dị iche iche recombinant spidroin protein (100 mg/mL) na 37 Celsius C na-enweghị ịma jijiji.Nnwale inoculation g NT (100 mg/mL) site na 100 mg/mL NT gel nwere 0%, 5%, 10%, na 20% mkpụrụ.
Nyocha nke gel site na iji microscopy eletrọnịkị nnyefe (TEM) gosipụtara na hydrogel nwere fibrils amyloid (Figs 3c, 3d).A na-agbatị fibrils nke NT-etolite (5-12 nm na dayameta) na enweghị alaka, ebe His-NT2RepCT fibrils dị mkpụmkpụ n'ogologo na nke ukwuu na dayameta (7-16 nm) (Fig 3e).Nsonaazụ ndị a nyere anyị ohere ịgbaso usoro kinetics nke fibrosis site na iji nyocha thioflavin T (ThT).Maka protein spidroin niile recombinant, mgbama fluorescent mụbara mgbe etinyere ihe nlele na 37 Celsius (Fig 3f, Fig. 5a mgbakwunye).N'ikwekọ na nchoputa a, nyocha microscopic nke NT na His-NT2RepCT n'okpuru ọnọdụ gelling kpughere otu mmụba nke ThT fluorescence na-enweghị ngosi mkpokọta mpaghara nke ThT-positive aggregates (Mgbakwunye Fig. 5b, c).Nhazi nke ThT-positive fibrils esoghị na mmụba nke NT na turbidity Ya-NTCT (Mgbakwunye 5d), nke pụtara na netwọk nke fibrils dị na gel nwere ike ịmalite na-enweghị imebi nchapụta gel.Ịgha mkpụrụ site n'ịgbakwụnye obere fibril emebere mbụ nwere ike ime ka fibril guzobe ụfọdụ amyloids42,43,44 mana na-agbakwunye 5%, 10% ma ọ bụ 20% (w/w) NT na ngwọta nke NT hydrocoagulants.mmetụta mkpụrụ (Fig 3g).Ikekwe nke a bụ n'ihi eziokwu ahụ bụ na fibrils dị na hydrogel dị ntakịrị na enweghị ike iji ya dị ka mkpụrụ.
Omume a na-atụghị anya ya nke protin spidroin recombinant na oke okpomọkụ mere ka ọmụmụ ihe nleba anya nke resonance nuklia (NMR) pụta iji chọpụta mgbanwe mgbanwe na-ejikọta na nhazi gel.NMR ụdịdị dị iche iche nke His-NT2RepCT ngwọta e dekọrọ na oge na 37 Celsius gosiri na CT ka na-apịaji akụkụ ụfọdụ, ebe NT na 2Rep akara n'anya (Fig. 4a), na-atụ aro na ọ bụ tumadi NT na 2Rep na-achịkwa akụkụ nke nhazi nke His- NT2RepCT hydrogel.A na-ebelata akara ngosi CT na 20% nke ike mbụ ya, na-atụ aro na CT na-ejikarị edozi ma tinye ya na nhazi hydrogel.Maka obere akụkụ nke CT, nke dị ka mkpanaka dị ka ọ dị na nlele preincubated ma si otú a na-ahụ ya site na ngwọta NMR, ụdịdị ahụ enweghị akara maka 10 mbụ a haziri ahazi, ikekwe n'ihi nkwụsị siri ike nke akụkụ nke His-NT2Rep.Ihe ngosi NMR nke steeti hydrogels -NT2RepCT gosipụtara ọnụnọ kachasị nke α-helices na β-layers na, n'ókè dị nta, nkwekọrịta mkpuchi mkpuchi na-enweghị atụ (Fig 4b).Ntụle mgbanwe kemịkalụ nke methionine residues dị naanị na NT gosiri na a gbanwere ngalaba a ka ọ bụrụ nhazi β-ibé akwụkwọ.Ụdị NT na-adabere na oge nke NT na ngwọta gosipụtara otu mbelata nke ike mgbaàmà (Fig 4c), na NMR siri ike nke NT hydrogels gosiri na ọtụtụ n'ime ihe ndị fọdụrụ na NT gbanwere na β-ibe akwụkwọ (Fig 4d).Enweghị ike ikpebi ngbanwe nke 2Rep iche iche n'ihi na ọ na-enwe ike ikpokọta.Otú ọ dị, ọnọdụ NMR siri ike nke NTCT na His-NT2RepCT hydrogels yiri nnọọ (Fig 4b; Mgbakwunye Fig. 6b), na-atụ aro na 2Rep nyere ntakịrị ihe na akụkụ nhazi nke His-NT2RepCT hydrogel.Maka CT hydrogels, α-helices, β-sheets, na usoro nke abụọ helical na-enweghị usoro ka achọpụtara dị (Ngbakwunye Fig. 6d).Nke a na-egosi na akụkụ ụfọdụ nke CT na-anọgide α-helices ebe ndị ọzọ na-aghọ β-ibé akwụkwọ.Ya mere, nsonaazụ nke NMR spectroscopy na-atụ aro na NT dị mkpa maka nhazi hydrogel ma na-agbanwekwa n'ime nkwekọrịta β-akwụkwọ n'elu ngwakọta na 2Rep na CT.N'ikwekọ na nke a, anyị chọpụtara n'oge na-adịbeghị anya na amyloid spatial zippers nwere ike ịmalite na helices ise nke ngalaba NT, na Waltz algorithm buru amụma na mpaghara amyloidogenic na helix 1 (Fig 4e).
2D ụdịdị 15N-HSQC 10 mg/mL His-NT2RepCT ngwọta tupu (acha anụnụ anụnụ) na 19 awa mgbe incubation (red) na 37°C.A na-eji akara amino acid nke otu mkpụrụedemede na ọnụọgụ fọduru na-akọwapụta ugwu obe nke otu onye na F24, G136, polyA na ụdị anụnụ anụnụ.Ntinye ahụ na-egosi ndabere nke ike mgbama ahụ na oge maka ihe fọdụrụ ahọpụtara sitere na ngalaba NT, 2Rep na CT.b Igwe ikuku redio siri ike (RFDR) nke His-NT2RepCT hydrogels.Nkwekọrịta nke ihe fọdụrụ Cα/Cβ hụrụ na RFDR spectra ka ekpebisiri ike site na ntụnyere ụdị mgbanwe kemịkalụ peptide na ụkpụrụ sitere na ọnụ ọgụgụ82,83 na ụlọ nke abụọ ha.SSB - eriri akụkụ na-atụgharị.c Otu akụkụ dị iche iche nke 15N-HSQC 10 mg/mL NT ngwọta n'oge incubation na 37 Celsius C maka awa 36.Ntinye ahụ na-egosi ike volumetric na oge.d Steeti siri ike RFDR ụdịdị NT hydrogels.Nkwekọrịta nke ihe fọdụrụ Cα/Cβ na usoro ha nke abụọ ahụrụ n'ụdị ụdị RFDR ka egosiri.e Dabere na profaịlụ fibrillation propensity NT45.79 sitere na nchekwa data Zipper (https://services.mbi.ucla.edu/zipperdb/).Ike Rosetta nke windo ngbanwe ọkụ nke hexapeptide ka egosiri na kcal/mol.Ogwe uhie na-egosi hexapeptides nwere nnukwu fibrosis propensity (Rosetta ike n'okpuru -23 kcal / mol; n'okpuru akara akara).Ogwe akwụkwọ ndụ akwụkwọ ndụ na-egosi iberibe nwere ike Rosetta n'elu ọnụ ụzọ ya mere o yikarịghị ka ọ ga-etolite zippers.E wepụrụ iberibe ihe ndị nwere proline na nyocha (na-enweghị ogidi).Square na-egosi mpaghara amyloidosis nke Waltz algọridim81 (https://waltz.switchlab.org) buru amụma ya.Usoro nke amino acid residues nke NT dị n'elu, a na-egosikwa ụdị ihe fọdụrụ n'ụdị nke abụọ β (nke siri ike steeti NMR spectroscopy kpebisiri ike) na-acha uhie uhie.Ọkwa nke ise NT α-helices ka ahapụtara dị ka (H1-H5)28.
Na pH <6.5, HT na-ebelata, na-eguzogide denaturation nke okpomọkụ ma ọ bụ urea na-ebute18.Iji kọwaa otú NT dimerization na nkwụsi ike si emetụta gelation, a na-achịkwa ihe ngwọta nwere 100 mg/ml NT na pH 8, 7, na 6 site na iji nyocha inversion vial.Ihe nlele NT nke etinyere na pH 8 na 7 gelled mgbe 30 min na 37 Celsius C, mana pH 8 gel nọgidere na-edo anya, ebe pH 7 gel gosipụtara ihe ngosi a na-ahụ anya (Fig 5a).N'ụzọ dị iche, ihe ngwọta nwere HT na pH 6 emepụtaghị gel, na nnukwu mmiri ozuzo nwere ike ịhụ mgbe 20 min na 37 Celsius C.Nke a na-egosi na dimers onwe ha na / ma ọ bụ nkwụsi ike ha dị elu ma e jiri ya tụnyere monomers na-egbochi gelation.A naghị atụ anya nhazi nke NT maka NT na pH 7 na 6, ebe ọ bụ na a kọwapụtara na NT bụ soluble na 200 mg / ml27, na-emegharị ngwa ngwa mgbe okpomọkụ denaturation, ma na-ejigide α-helix na ọnụ ala dị ala. pH 18. Nkọwa enwere ike maka ọdịiche ndị a bụ na a na-eme nnwale ndị a kọrọ na mbụ na ọnụ ụlọ okpomọkụ ma ọ bụ n'okpuru, ma ọ bụ na protein dị ala16,18,19.
Nnwale inversion nke NT vial (100 mg/mL) na pH 8, 7, 6 na 154 mM NaCl (pH 8) ka emechara ya na 37°C.b NT CD ụdịdị nke nwere na enweghị 154 mM NaF na 154 mM NaCl, n'otu n'otu.A na-atụgharị elliptical molar na 222 nm ka ọ bụrụ oke nke mpịaji eke.c NT inversion assay (100 mg/mL) NT * (37 Celsius C na 60 Celsius C), NTA72R (37 Celsius C), na His-NT-L6 (37 Celsius C na 60 Celsius C).d ụdịdị CD nke NT mutant NT*, NTA72R, na His-NT-L6.A na-atụgharị elliptical molar na 222 nm ka ọ bụrụ oke nke mpịaji eke.e Nnwale ntụgharị nke NTFLSp, NTMiSp na NTMiSp belatara (100 mg/mL).Oghere ọnụ ọgụgụ 5 mm.f ụdịdị CD nke NT, NTFLSp, NTMiSp yana NTMiSp belatara.A na-atụgharị elliptical molar na 222 nm ka ọ bụrụ oke nke mpịaji eke.Eserese NT zuru oke na 25 Celsius na 95 Celsius ka egosiri na foto mgbakwunye 8.
Nleba nnu nke anụ ahụ na-ekpebi mmekọrịta electrostatic n'etiti NT subunits na dimerization nke mbufe NT na pH18 dị ala.Anyị chọpụtara na ọnụnọ nke 154 mM NaCl na NaF mere n'ezie igbochi gelation, n'otu n'otu (Fig. 5a, b; Ihe mgbakwunye 2b) nakwa na nnu ndị a na-eme ka nkwụsi ike nke okpomọkụ nke NT monomers (Fig. 5b, Fig. 8). .Ọ na-atụkwa aro na nkwalite nkwụsi ike, kama ịbelata, na-egbochi nhazi gel.
Iji chọpụtakwuo ọrụ nke dimerization protein na nkwụsi ike na gelation, anyị na-eji mutant abụọ, NT * na NTA72R, nke na-anọgidekwa na-enwe monomeric na obere pH28.30.NT * bụ mutant ngbanwe ụgwọ okpukpu abụọ nke nkesa nkesa dipolar nke monomer na-adaba adaba, nke na-egbochi dimerization ma na-abawanye nkwụsi ike nke monomer.NTA72R bụ dipole ebubo, mana Arg-dochiri Ala dị na ókèala dimer, yabụ ngbanwe na-egbochi mmekọrịta nke subunit chọrọ maka ngbanwe.Mgbe incubation na 37 Celsius C, NT * emeghị hydrogel, ebe NTA72R na-emepụta gel opaque maka 15 min (Fig 5c).Ebe ọ bụ na ma NT * na NTA72R enweghị ike ibelata ma dị iche na nkwụsi ike nke monomer (Fig 5d), nsonaazụ ndị a na-egosi na nkwụsi ike nke thermodynamic dị elu na-egbochi NT site na gelling.Nke a na-akwadokwa eziokwu ahụ bụ na HT * na-emepụta gel mgbe ọ na-ejighị n'aka na okpomọkụ dị elu (mgbe 8 min na 60 ° C; Fig. 5c).E gosipụtara na mbụ na nnukwu ọdịnaya nke methionine dị na NT na-eme ka mpịakọta eke ya na isii Met ka Leu dochie anya (nke a na-akpọ ebe a dị ka Ya-NT-L6) na-eme ka monomer NT46 sie ike.Dabere n'echiche ahụ na a chọrọ mgbanwe nhazi maka nhazi NT gel, anyị chọpụtara na mutant nke His-NT-L6 emeghị ka ọ dị na 37 Celsius C (Nyocha 5c, d).Otú ọ dị, His-NT-L6 mekwara gel n'elu incubation na 60 Celsius maka 60 min (Fig. 5c).
Ikike nke NT ịtụgharị gaa n'ụdị β-sheet na imepụta hydrogels yiri ka ọ na-emetụta ụfọdụ mana ọ bụghị ngalaba NT niile nke spidroin.NT sitere na ụdị silk dị iche iche na ụdị ududo, Trichonephila clavipes (NTFlSp), gels kpụrụ n'agbanyeghị ọdịnaya methionine dị ntakịrị na nkwụsi ike dị elu (Fig 5e, f na tebụl mgbakwunye 2).N'ụzọ dị iche, NT si na obere spidroin protein ampullar sitere na Araneus ventricosus (NTMiSp) nwere nkwụsi ike dị ala na ọdịnaya methionine dị elu emepụtaghị hydrogels (Mgbakwunye 2 na Fig. 5e, f).Enwere ike jikọta nke ikpeazụ na ọnụnọ nke intramolecular disulfide bonds29,47.Na-adịgide adịgide, mgbe a na-ebelata njikọ disulfide nke NTMiSp, ọ na-emepụta hydrogel mgbe a na-etinye ya na 37 ° C maka 10 min (Fig 5e).N'ikpeazụ, ekwesịrị iburu n'uche na mgbanwe nhazi bụ ihe dị mkpa, ma ọ bụghị naanị, njirimara maka ịmepụta gel sitere na NT.Ihe ọzọ nwere ike ịdị mkpa bụ ọchịchọ ịmepụta amyloid fibrils, na nyocha na nchekwa data zipa na Waltz algorithm gosipụtara njikọ dị n'etiti ikike ịmepụta gels na ọnụnọ nke mpaghara amyloidogenic, yana oke mpaghara ndị e buru n'amụma. iji mepụta zippers stric.Enwere njikọ (Table 2 na mgbakwunye Fig. 9).
Ikike nke NT ịmepụta fibrils na ịmepụta gels n'okpuru ọnọdụ dị mma mere ka anyị chee na NT fusions na protein ndị ọzọ nwere ike ịmepụta gels nwere ọrụ zuru oke nke ndị mmekọ njikọ.Iji nwalee nke a, anyị webatara protein fluorescent green (GFP) na purine nucleoside phosphorylase (PNP) na C-terminus nke NT, n'otu n'otu.E gosipụtara protein fusion nke si na ya pụta na E. coli nwere mkpụrụ ikpeazụ dị elu (150 mg/L na 256 mg/L shake flask cultures maka His-NT-GFP na His-NT-PNP, n'otu n'otu), kwekọrọ n'ihe egosiri. maka protein ndị ọzọ jikọtara na NT Ref.30. His-NT-GFP (300mg / mL) na His-NT-PNP (100mg / mL) protein fusion kpụrụ gels mgbe 2 awa na 6.5 awa na 37 ° C na, dị mkpa, GFP ụmụ irighiri nọgidere na-agbanweghi agbanwe.hụrụ mgbe gelation gasịrị, na> 70% nke mmalite fluorescence ike fọdụrụ mgbe gelation (Fig. 6a).Iji tụọ ọrụ PNP na ngwọta ya-NT-PNP na gels, anyị ga-eji NT mee ka protein fusion jupụta n'ihi na ọrụ enzymatic nke nkwadebe dị ọcha dị n'èzí nchọpụta nke nyocha ahụ na mkpokọta gelling.Gel a kpụrụ na ngwakọta nwere 0.01 mg/mL His-NT-PNP na 100 mg/mL NT jigidere 65% nke mbụ enzymatic ọrụ nke preincubated samples (Fig. 6b).Gel nọgidere na-emebi emebi n'oge nha (Fig 10 mgbakwunye).
a ikwu fluorescence ike tupu na mgbe gelation nke His-NT-GFP (300 mg/mL) na tụgharịa vial nwere His-NT-GFP hydrogel (300 mg/mL) n'okpuru anya na UV ìhè.Isi ihe na-egosi nha n'otu n'otu (n = 3), ogwe mperi na-egosi ngbanwe ọkọlọtọ.Egosiri nkezi uru n'etiti ogwe njehie.b PNP nwetara site na nyocha nke fluorometric site na iji ngwọta na gels nwere NT (100 mg / ml) na ngwakọta nwere 0.01 mg / ml his-NT-PNP na 100 mg / ml dollar Taiwan ọhụrụ.Ntinye ahụ na-egosi vial tụgharịrị nke nwere hydrogel nwere His-NT-PNP (ogwe akpịrịkpa 5 mm).
N'ebe a, anyị na-akọ na e guzobere hydrogels sitere na NT na ndị ọzọ na-edozi spidroin protein site na incubating protin ngwọta na 37 ° C (Figure 1).Anyị na-egosi na gelation jikọtara ya na mgbanwe nke α-helices n'ime β-layers na nhazi nke amyloid dị ka fibrils (Figs 3 na 4).Nchọpụta a bụ ihe ijuanya dị ka NTs na-agbakọta globular globular-helix bundles mara maka oke solubility ha na nkwụsi ike dị elu na ntinye> 200 mg / ml na 4 ° C ruo ọtụtụ ụbọchị27.Na mgbakwunye, NTs na-agbagharị ngwa ngwa mgbe denaturation okpomọkụ gasịrị na mkpokọta protein dị ala na µM.Dị ka nsonaazụ anyị si dị, nhazi fibril chọrọ nchikota> 10 mg / mL protein ntinye uche na obere okpomọkụ dị elu (Fig 1).Nke a kwekọrọ n'echiche ahụ na fibrils amyloid nwere ike ịmalite site na protein ndị na-agbaji globularly nke dị n'ụdị nke na-emepe emepe n'ihi mgbanwe okpomọkụ n'okpuru ọnọdụ physiological 48.Ihe atụ nke protein ndị na-eme mgbanwe a gụnyere insulin49,50, β2-microglobulin, transthyretin na lysozyme51,52,53.Ọ bụ ezie na NT bụ α-helix na ala ala nna ya, ihe dịka 65% nke eriri polypeptide dakọtara na nhazi nke stric zipper (Fig 4e) 45.Ebe ọ bụ na monomer bụ mobile dynamically46, ọ nwere ike ikpughe mpaghara amyloidogenic ndị a na oke okpomọkụ dị elu yana n'ogo dị elu nke protein zuru oke nwere ike iru n'ọkwa dị egwu maka nhazi amyloid fibril54.N'ịgbaso echiche a, anyị chọtara mmekọrịta na-adịghị mma n'etiti mkpokọta spidroin na oge gelation (Fig 1c), ma ọ bụrụ na monomeric NT conformation kwadoro ma ọ bụ site na ngbanwe (NT *, His-NT-L6) ma ọ bụ site na mgbakwunye nnu, nwere ike igbochi ya. nhazi hydrogels (Fig 5).
N'ọtụtụ ọnọdụ, amyloid fibrils na-apụ n'anya na ngwọta dị ka mmiri ozuzo, ma n'ọnọdụ ụfọdụ ha nwere ike ịmepụta hydrogels55,56,57.Fibril na-akpụ hydrogel na-enwekarị akụkụ dị elu ma na-etolite netwọkụ nwere akụkụ atọ kwụsiri ike site na njikọ molecular, 55,58 kwekọrọ na nsonaazụ anyị.Maka nhazi hydrogel in vitro, protein na-apụtakarị n'ụzọ zuru ezu ma ọ bụ akụkụ ụfọdụ, dịka ọmụmaatụ, site na ikpughe na ihe mgbaze organic, okpomọkụ dị elu (70-90 ° C) na / ma ọ bụ pH dị ala (1.5-3.0) 59,60,61,62.Ndị spidroin hydrogels akọwara ebe a adịghị achọ nhazi siri ike, ma ọ bụ na ha achọghị ndị na-ejikọta ọnụ iji mee ka hydrogels guzosie ike.
A kọwo na mbụ na spidroin na-emegharị na QDs, nke yiri ka ọ na-agbanwe β-sheet n'oge a na-atụgharị silk, na-etolite hydrogels.E jiri ya tụnyere nchoputa anyị, oge ntinye na / ma ọ bụ ikpo ọkụ na-adị ogologo ma ọ bụ karịa, n'otu n'otu, na hydrogels ndị na-esi na ya pụta na-abụkarị opaque (Figure 7 na Tebụl mgbakwunye 1) 37, 38, 63, 64, 65, 66, 67, 68 , 69. Na mgbakwunye na oge gel ngwa ngwa, NT hydrogels> 300 mg / mL (30%) karịrị ihe niile a kọwara recombinant ududo silk protein hydrogels, yana hydrogels eke dị ka gelatin, alginate (2%), agar (0.5%). ) na collagen.(0.6%) (Foto 7 na Tebụl mgbakwunye 1 na 3)37,39,66,67,68,69,70,71,72,73,74.
Atụnyere oge gel na modulus nke hydrogels n'ime ọmụmụ a na hydrogels ndị ọzọ dabere na spidroin yana hydrogels eke ahọpụtara.A na-enye ntụaka yana nkọwa nke ọnọdụ gelation.APS Ammonium persulfate, ọnụ ụlọ okpomọkụ.Data 37, 38, 39, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
Ududo na-egosi na ha mepụtara ụzọ iji gbochie spidroin site na gelling n'oge nchekwa.N'agbanyeghị oke protein dị na gland silk, nnukwu mpaghara ikwugharị na-ejikọta na njedebe njedebe pụtara na ntinye anya nke NT na CT dị na gland ahụ kwekọrọ na 10-20 mg / ml, na njedebe nke ọmụmụ a.achọrọ maka nhazi hydrogel in vitro hụrụ.Ke adianade do, yiri ịta nke salts 16 kwụsie ike NT, dị ka silk glands (Fig. 5b).A mụọla NT conformation na E. coli cytosol wee chọpụta na ọ na-apịaji karịa mgbe a na-enyocha ya na vitro, na-egosikwa na nnu ma ọ bụ ihe ndị ọzọ na-egbochi nchịkọta ya na vivo.Otú ọ dị, ikike nke NT iji gbanwee n'ime β-sheet fibrils nwere ike ịdị mkpa maka ịmepụta filament na ekwesịrị nyochaa ya na ọmụmụ ihe n'ọdịnihu.
Na mgbakwunye na akụkụ akwụkwọ akụkọ nke NT-amyloid-dị ka fibril na hydrogel formation hụrụ na ọmụmụ a, anyị na-egosikwa na ihe omume a nwere ike ịnwe ngwa biotechnological na biomedical (Fig 8).Dị ka ihe akaebe nke echiche, anyị jikọtara NT na GFP ma ọ bụ PNP wee gosi na protein fusion na-emepụtakwa hydrogels mgbe a na-etinye ya na 37 Celsius C yana na GFP na PNP fractions na-ejigide ọrụ ha mgbe gelation gasịrị (Nyocha 6).Nucleoside phosphorylases bụ ihe dị mkpa na-eme ka njikọ nke nucleoside analogues75, nke na-eme ka nchọpụta anyị dị mkpa maka ụlọ ọrụ biopharmaceutical.Echiche nke ịkọwapụta protein fusion nke na-etolite hydrogels transperent n'okpuru ọnọdụ dị mma na-enye ohere ịmepụta hydrogels na-arụ ọrụ nke ọma na ihe ndị dị mma maka ọtụtụ ngwa dị ka enzyme immobilization, nkwụsị ọgwụ na-achịkwa na nhazi anụ ahụ.Na mgbakwunye, NT na NT * bụ akara ngosipụta nke ọma30, nke pụtara na enwere ike iji NT na ụdị ya dị iche iche maka nrụpụta dị elu nke protein fusion soluble yana mechaa mepụta protein ndị ebu n'obi na-enweghị ike ime na 3D hydrogels.
NT bụ soluble, α-helical na kwụsiri ike na obere mkpokọta (µM) na 37°C.N'otu okpomọkụ ahụ, ma na-abawanye ụba (> 10 mg / ml), NT na-emepụta gels nke nwere amyloid-dị ka fibrils.Ndị na-edozi NT fusion na-etolitekwa gels fibrillar nwere iberibe ngwakọta na-arụ ọrụ zuru oke, na-ekwe ka protein dị iche iche ghara imegharị na 3D hydrogels site na iji NT.N'okpuru: NT (PDB: 4FBS) na ihe atụ nke netwọọdụ eriri na usoro protein jikọtara ya (nke a na-ewere na ọ bụghị nke a na-adọta na ọnụ ọgụgụ, GFP PDB: 2B3Q, 10.2210/pdb2B3Q/pdb; PNP PDB: 4RJ2, 10.2210/2/pdb4).
Ihe arụrụ arụ (lee tebụl mgbakwunye 4 maka ndepụta zuru oke gụnyere usoro amino acid) ka etinyere ka ọ bụrụ plasmid pT7 wee gbanwee ghọọ E. coli BL21 (DE3).E. coli nwere plasmids injinia ka etinyere na broth Luria gbakwunyere na kanamycin (70 mg / l) wee tolite n'abali na 30 Celsius C na 250 rpm.A na-etinyekwa omenala ahụ 1/100 n'ime usoro LB nwere kanamycin ma mee ya na 30°C na 110 rpm ruo mgbe OD600 ruru 0.8.Maka ọmụmụ NMR, nje bacteria na-etolite na M9 minimal medium nwere 2 g nke D-glucose 13C (Aldrich) na 1 g nke ammonium chloride 15N (Cambridge Isotope Laboratories, Inc.) maka akara protein na isotopes.Wetuo okpomọkụ ruo ogo Celsius 20 wee weta okwu protein na 0.15 mM isopropylthiogalactopyranoside (ntụkwasị obi ikpeazụ).Mgbe nkwupụta protein gasịrị, a na-egbute mkpụrụ ndụ na 7278 × g, 4 Celsius C maka nkeji 20.A kwụsịrị pellets cell na 20 mM Tris-HCl, pH 8, na oyi kpọnwụrụ ruo mgbe ejiri ya ọzọ.A na-ekpuchi mkpụrụ ndụ thawed site na iji ihe na-emebi cell (igwe usoro TS, Constant Systems Limited, England) na 30 kPa.Mgbe ahụ, a na-agbanye lysates na 25,000 g maka nkeji 30 na 4 Celsius.Maka NTMiSp, eweghachiri pellet ahụ na 2 M urea, 20mM Tris-HCl, pH 8, na sonicated maka 2 min (2 s gbanyụọ, 65%), wee centrifuged ọzọ na 25,000 xg, 4° C. n'ime 30 min.A na-ebunye ihe dị elu na kọlụm Ni-NTA, jiri 20 mM Tris-HCl, 2 mM imidazole, pH 8, na n'ikpeazụ, e ji 20 mM Tris-HCl, 200 mM imidazole, pH 8. Iji mepụta NT2RepCT na protein. NTCT, mgbaze thrombin na-ewebata saịtị (ThrCleav) n'etiti Ya na NT.Ebe ikpochapu Thrombin dịkwa na His-NT-ThrCleav-2Rep (na-emepụta 2Rep), His-thioredoxin-ThrCleav-NT (na-emepụta NT), His-thioredoxin-ThrCleav-CT (na-emepụta CT), His-Thioredoxin-ThrCleav-NT. .* (na-emepụta NT *), His-Thioredoxin-ThrCleav-NTA72R (na-emepụta NTA72R), His-Thioredoxin-ThrCleav-NTFlSp (na-emepụta NTF1Sp), na His-Sulphur Redoxin-ThrCleav-NTMiSp (na-emepụta NTMiSp).Ejiri thrombin (1: 1000) gbarie ihe ndị a na-arụ ma mee ya n'otu abalị na 4 ° C. na 20 mM Tris-HCl, pH 8, na-eji akpụkpọ anụ Spectra / Por dialysis nwere ọnụ ụzọ molecular arọ nke 6-8 kDa.Mgbe dialysis gasịrị, a na-etinye ihe ngwọta n'elu kọlụm Ni-NTA ma na-anakọta nsị nwere protein nke mmasị.Achọpụtara mkpokọta protein site na ịlele nnabata UV na 280 nm site na iji ọnụọgụ mbibi nke protein ọ bụla, ewezuga NTF1Sp, nke jiri nyocha Bradford dị ka ụkpụrụ ndị nrụpụta siri dị.SDS polyacrylamide (4–20%) gel electrophoresis na Coomassie na-acha anụnụ anụnụ kpebisiri ịdị ọcha.Etinyere protein n'iji ihe nzacha centrifuge (VivaSpin 20, GE Healthcare) na 4000 xg nwere mbelata ịdị arọ molekụla 10 kDa n'ime nkeji nkeji iri abụọ.
Ghichaa ngwọta protein ma jiri nlezianya kpoo 150 µl n'ime vial septum doro anya 1 ml (8 x 40 mm Thermo Scientific).A na-ekpuchi akpa ndị ahụ ma jiri parafilm mechie ya ka ọ ghara ikpochapụ.A na-etinye ihe nlele (n = 3) na 37°C ma ọ bụ 60°C ma na-atụgharị kwa oge iji hụ gelation.A na-etinye ihe nlele nke na-abụghị gel maka opekata mpe otu izu.Belata njikọ NTMiSp disulfide na 10 mM DTT kwa protein 10 µM.Iji nyochaa gelation nke mkpuchi silk ududo eke, a na-egbutu ududo ududo nke Sweden, a na-etinye isi ihe abụọ a na-egbutu na 200 μl nke 20 mM Tris-HCl buffer pH 8 ma bee ka mkpuchi ahụ kewapụ na glands..A na-agbaze ọdịnaya nke glands na nchekwa, 50 µl maka ikpebi ịdị arọ akọrọ (site na ntinye nke vials na-emeghe na 60 Celsius C ruo mgbe ịdị arọ) na 150 µl maka gelation na 37 Celsius.
A na-eji igwe anaghị agba nchara mee ihe jiometrị / ngwá ọrụ na-eji efere yiri ya na dayameta dị elu nke 20 mm na ọdịiche nke 0.5 mm.Kpoo ihe nlele ahụ site na 25 Celsius C ruo 45 Celsius C wee laghachi na 25 Celsius C na ọnụego 1 Celsius C kwa nkeji site na iji igwe anaghị agba nchara na ala Peltier efere.A na-eme ihe nlegharị anya na ugboro 0.1 Hz na mpaghara viscoelastic linear nke ihe dị na 5% na 0.5% maka ihe atụ nke 100 mg / mL na 300-500 mg / ml, n'otu n'otu.Jiri ụlọ iru mmiri nke omenala iji gbochie evaporation.A tụlere data site na iji Prism 9.
Maka ịnakọta ihe nkiri infrared (IR) na ụlọ okpomọkụ site na 800 ruo 3900 cm-1.A na-eji ikuku akọrọ kpochaa ngwaọrụ ATR, yana ụzọ ọkụ si na spectrometer, tupu na n'oge nnwale ahụ.Ngwọta (500 mg / mL iji belata ọnụ ọgụgụ mmịpụta mmiri na spectra) bụ ọkpọkọ na kristal, na gels (500 mg / mL) na-emepụta tupu nha wee bufee ya na kristal (n = 3).Edere nyocha 1000 na mkpebi nke 2 cm-1 na okirikiri ọrụ efu nke 2. A na-agbakọ ihe nrụpụta nke abụọ site na iji OPUS (Bruker) site na iji mgbatị dị nro nke isi itoolu.A na-ahazi ihe nkiri ahụ ka ọ bụrụ otu mpaghara njikọta n'etiti 1720 na 1580 cm-1 site na iji F. Menges "Spectragryph - Optical Spectroscopy Software".Na ATR-IR spectroscopy, omimi ntinye nke infrared beam n'ime ihe nlele na-adabere na ọnụọgụ ebili mmiri, na-ebute mmịnye siri ike na ebili mmiri dị ala karịa na ọnụọgụ ebili mmiri dị elu.Emezibeghị mmetụta ndị a maka ụdịdị dị na fig.3 n'ihi na ha dị nnọọ obere (Nkọwa Fig. 4).Ejiri sọftụ Bruker OPUS gbakọọ ihe onyonyo emeziri maka ọnụ ọgụgụ a.
N'ụkpụrụ, ọnụ ọgụgụ zuru oke nke nhazi protein ga-ekwe omume mgbe a pụrụ ịdabere na ya deconvolution nke ihe ndị dị n'ime amide I peak.Agbanyeghị, ụfọdụ ihe mgbochi na-ebilite na omume.Mkpọtụ dị na ụdịdị dị iche iche nwere ike ịpụta dị ka ọnụ ọgụgụ kacha elu (ụgha) n'oge mgbanwe mgbanwe.Tụkwasị na nke ahụ, ọnụ ọgụgụ kasị elu n'ihi mmiri na-ehulata na-adaba na ọnọdụ nke amide I peak ma nwee ike inwe oke yiri nke ahụ maka ihe atụ nwere nnukwu mmiri, dị ka gel aqueous nke a na-amụ ebe a.Ya mere, anyị anwaghị imebi oke amide I zuru oke, na ihe anyị kwuru kwesịrị ka a tụle ya na nkwado nke ụzọ ndị ọzọ dị ka NMR spectroscopy.
Ngwọta nke 50 mg/ml NT na His-NT2RepCT na-agbaze n'otu abalị na 37 Celsius.A na-agbanye hydrogel na 20 mM Tris-HCl (pH 8) na ntinye nke 12.5 mg / ml, maa jijiji nke ọma na pipetted iji mebie gel.Na-esote, a na-agbaze hydrogel ugboro 10 na 20 mM Tris-HCl (pH 8), 5 μl nke ihe nlele ahụ na-etinye ya na grid ọla kọpa nke ejiri formvar kpuchie ya, ma wepụ ihe nfefe ahụ na mpempe akwụkwọ.Eji 5 µl nke mmiri MilliQ saa ihe nlele ugboro abụọ ma jiri 1% uranyl formate mee ntụ maka nkeji 5.Jiri akwụkwọ na-agbapụta ihe were wepu unyi ngafe, wee kpochaa ntupu ahụ.Emere onyonyo na grid ndị a site na iji FEI Tecnai 12 Spirit BioTWIN na-arụ ọrụ na 100 kV.Edere onyonyo a na x 26,500 na x 43,000 magnifications site na iji igwefoto Veleta 2k × 2k CCD (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).Maka nlele ọ bụla (n = 1), ihe oyiyi 10-15 dekọrọ.A na-eji ImageJ (https://imagej.nih.gov/) mee ihe maka nyocha onyonyo na nha nke dayameta eriri (n = 100, eriri dị iche iche).A na-eji Prism 9 mee t-ule na-enweghị atụ (ọdụdụ abụọ).Ihe pụtara His-NT2RepCT na NT fibrils bụ 11.43 (SD 2.035) na 7.67 (SD 1.389) nm, n'otu n'otu.Oge ntụkwasị obi (95%) bụ -4.246 ruo -3.275.ogo nnwere onwe = 198, p <0.0001.
80 µl nke ihe nlele mmiri nwere 10 µM thioflavin T (ThT) tụrụ na triplicate (n = 3) n'okpuru ọnọdụ static site na iji Corning 96-nke ọma ojii ala doro anya efere ala (Corning Glass 3881, USA).Edere ọdịiche dị iche iche nke fluorescence site na iji nzacha mkpali 440 nm yana nzacha nm 480 (FLUOStar Galaxy sitere na BMG Labtech, Offenburg, Germany).Ejughi akara ngosi ThT ma ọ bụ menyụọ, ebe a na-eme nnwale ndị nwere nleba anya dị iche iche nke ThT na-agbanweghị ike mgbama.Dekọọ nnabata na 360nm maka nha haze.Maka nnwale mkpụrụ, 100 mg/mL gels ka e hibere na 37 Celsius C., kwụsịtụrụ, ma jiri ya mee ihe maka ịgha mkpụrụ na oke molar nke 5%, 10%, na 20%.A tụlere data site na iji Prism 9.
Kpoo ebuka nke His-NT2RepCT na NT>100 mg/mL na ice wee kpochaa site na nzacha 0.22µm.A gbakọrọ nlebara anya site n'ịtụ nnabata na 280 nm site na iji Nandrop.N'ime olulu mmiri nke efere 96 nke oji na-adịghị ejikọta (Corning) nke nwere ala doro anya, a na-agbanye ihe nlele na 20 mg / ml na 20 mM Tris-HCl pH 8 ma jikọta ya na 5 μM ThT (ntụgharị ikpeazụ), mkpokọta mkpokọta mkpokọta. 50 μl olu.A na-esetịpụ ihe nlele na nkeji iri ọ bụla na 37 Celsius na microscope CellObserver (Zeiss) nwere ọwa ọkụ na-ebufe yana ihe nzacha FITC na ihe nzacha maka onyonyo ThT.A na-eji oghere 20x/0.4 maka eserese.Ejiri Zen Blue (Zeiss) na ImageJ (https://imagej.nih.gov/) mee ihe maka nyocha onyonyo.A kwadoro gels site na ngwọta NT na His-NT2RepCT na ntinye nke 50 mg/mL nwere 20 mM Tris pH 8 na 5 µM ThT ma tinye ya na 37 ° C maka 90 min.A na-ebufe iberibe gel ahụ na olulu mmiri ọhụrụ nwere 20 mM Tris, pH 8, na 5 μM ThT n'ime efere ojii 96 na-adịghị ejikọta nke ọma.Nweta fluorescence na-acha akwụkwọ ndụ akwụkwọ ndụ na onyonyo ubi na-egbuke egbuke na mmụba 20x/0.4.Ejiri ImageJ mee nyocha ihe onyonyo.
Ngwọta NMR spectra enwetara na 310 K na 600 MHz Bruker Avance Neo spectrometer nke nwere QCI Quadrupole Resonance Pulsed Gradient Field Cryoprobe (HFCN).Ihe nlele NMR nwere protein 10 mg/mL nke akpọrọ aha ya na 13C, 15N, gbazere na 20 mM Tris-HCl (pH 8), 0.02% (w/v) NaN3, 5% DO (v/v), (n = 1) .A na-eji mgbanwe kemịkalụ nke NT2RepCT na pH 6.7 kenye ọnụ ọgụgụ kasị elu 23 na ụdịdị 2D nke 15N-HSQC.
Edekọbara akụkụ anwansị siri ike nke NMR (MAS) nke 13C, hydrogels akara 15N na Bruker Avance III HD spectrometer na 800 MHz nwere nyocha 3.2 mm 13C/15N{1H} na-enweghị elektrọn.A na-achịkwa ihe atụ nke okpomọkụ site na iji mmiri gas na-agbanwe agbanwe na 277 K. Abụọ-dimensional dipole rotational resonance (DARR) 76 na redio reconnection (RFDR) 77 spectra ka enwetara na ugboro MAS nke 12.5 kHz na 20 kHz, n'otu n'otu.Cross polarization (CP) sitere na 1H ruo 13C mere site na iji eriri ahịrị site na 60.0 ruo 48.0 kHz na 1H, 61.3/71.6 kHz na 13C (na 12.5/20 kHz MAS) na oge kọntaktị 0.5-1 ms.Ejiri Spinal6478 decoupling na 73.5 kHz mee ihe n'oge nchịkọta data.Oge nnweta bụ milisekọnd 10 yana igbu oge okirikiri bụ 2.5 sekọnd.A na-ekenye njikọ Cα/Cβ nwere otu njikọ a na-ahụ na spectra RFDR dabere na njirimara mgbanwe kemịkalụ nke fọdụrụ na-ejikọta ya na ọtụtụ njikọ jikọtara na spectra DARR.
A na-eji nchekwa data Zipper79 (https://services.mbi.ucla.edu/zipperdb/) iji nyochaa ntule na ike Rosetta maka NT, NTFLSp, na NTMiSp.Ebe nchekwa data Zipper na-agbakọ Rosetta Energy80, nke na-ejikọta ọtụtụ ọrụ ume efu iji ṅomie na nyochaa nhazi protein.Ọkwa ike nke -23 kcal / mol ma ọ bụ nke dị ala na-egosi ọchịchọ dị elu nke fibrilate.Ike dị ala pụtara nkwụsi ike karịa nke eriri abụọ β-eriri na conformation zipa.Na mgbakwunye, ejiri Waltz algọridim mee amụma mpaghara amyloidogenic na NT, NTFLSp na NTMiSp Ref.81. (https://waltz.switchlab.org/).
Ejikọtara ihe ngwọta protein NT na 2- (N-morpholino) ethanesulfonic acid (MES) na pH 5.5 na 6.0 iji wedata pH ruo pH 6 na 7, n'otu n'otu.Ntụle protein ikpeazụ bụ 100 mg / ml.
A na-eme nha na J-1500 CD spectrometer (JASCO, USA) site na iji cuvette 300 μL nwere ụzọ anya nke 0.1 cm.A na-agbanye protein na 10 μM (n = 1) na 20 mM phosphate buffer (pH 8).Iji nyochaa nkwụsi ike protein na ọnụnọ nnu, a na-enyocha protein n'otu ntinye uche (n = 1) na 20 mM phosphate buffer (pH 8) nke nwere 154 mM NaF ma ọ bụ NaCl, n'otu n'otu.A na-edekọ nyocha okpomọkụ na 222 nm site na 25 ° C ruo 95 ° C na ọnụego ọkụ nke 1 ° C / min.A gbakọrọ ọnụ ọgụgụ nke protein ndị apịaji n'ala site na iji usoro (KDmeasure - KDfinal)/(KDstart - KDfinal).Tụkwasị na nke ahụ, a na-edekọ ihe ngosi ise maka nlele ọ bụla site na 260 nm ruo 190 nm na 25 ° C na mgbe ikpo ọkụ ruo 95 ° C.A na-agbakọ ụdịdị ise dị iche iche, mee ka ọ dị nro ma gbanwee ka ọ bụrụ elliptical molar.A tụlere data site na iji Prism 9.
A tụrụ ike fluorescence nke His-NT-GFP (300 mg/mL, 80 µL) na triplicate (n = 3) na 96-well Corning plates nwere ala transperent ojii (Corning Glass 3881, USA) n'okpuru ọnọdụ kwụ ọtọ.Jiri ihe na-agụ efere dabere na fluorescence nwere ogologo oge mkpali nke 395 nm wee dekọọ ihe mgbapụta ahụ na 509 nm tupu gelation na awa 2 gachara na 37°C.Eji Prism 9 nyochaa data.
A na-eji purine nucleoside phosphorylase ọrụ assay kit (usoro fluorometric, Sigma Aldrich) dịka ntuziaka onye nrụpụta si dị.Iji tụọ ọrụ na gels na ngwọta nwere His-NT-PNP, gwakọta 10 ng nke His-NT-PNP na 100 mg/mL NT na mkpokọta 2 µL n'ihi na gel nyere akara n'elu oge nchọpụta nke setịpụrụ.Agụnyere njikwa maka gels na ngwọta na-enweghị His-NT-PNP.A na-eme nha ahụ ugboro abụọ (n = 2).Mgbe a tụlechara ọrụ ahụ, ewepụrụ ngwakọta mmeghachi omume ahụ wee see foto gel iji hụ na gel ahụ dịgidere n'oge nha.A tụlere data site na iji Prism 9.
Maka ozi ndị ọzọ gbasara nhazi ọmụmụ, hụ ihe ọmụmụ ihe okike jikọtara na edemede a.
Ọnụọgụ 1 na 2 na-egosi data mbụ.1c, 2a–c, 3a, b, e–g, 4, 5b, d, f, na 6, fig mgbakwunye.3, fig mgbakwunye.5a, d, fig mgbakwunye.6 na mgbakwunye fig.8. A na-akwado data sitere na ọmụmụ ihe na nchekwa data Zenodo https://doi.org/10.5281/zenodo.6683653.E zigara data NMR enwetara na ọmụmụ a na ebe nchekwa BMRBig n'okpuru ntinye ID bmrbig36.Ewere ihe owuwu nke GFP na PNP sitere na PDB (GFP 2B3Q, PNP 4RJ2).
Na-ebili, A. na Johansson, J. Na-agbagharị silk udide artificial.National Chemical.bayoloji.11, 309–315 (2015).
Babb, PL et al.Nephila clavipes genome na-akọwapụta ụdị dị iche iche nke mkpụrụ ndụ ihe nketa silk udide na okwu mgbagwoju anya ha.National Genette.49, 895–903 (2017).