310 10 * 1mm Stainless hlau coiled tubing tshuaj tivthaiv, N-terminal domains ntawm spidroin daim ntawv hydrogels raws li amyloid fibrils thiab muab lub platform rau protein immobilization.

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Specification

310 10 * 1mm Stainless hlau coiled tubing lwm tus neeg

Tshuaj muaj pes tsawg leeg

Mechanical Properties

Recombinant kab laug sab silk proteins (kab laug sab txhob lo lo ntxhuav proteins) muaj ntau yam muaj peev xwm siv nyob rau hauv txoj kev loj hlob ntawm biomaterials tshiab, tab sis lawv multimodal thiab aggregation-nrhiav qhov ua rau lawv nyuaj rau tau thiab yooj yim rau siv.Ntawm no peb tshaj tawm tias cov khoom me me ntawm cov kab laug sab spidroin recombinant thiab, qhov tseem ceeb, N-terminal domain (NT) nws tus kheej sai sai tsim tus kheej txhawb nqa thiab pob tshab hydrogels ntawm 37 ° C.fusion proteins uas muaj NT thiab ntsuab fluorescent protein los yog purine nucleoside phosphorylase tsim ua haujlwm fusion proteins.Hydrogels.Peb cov txiaj ntsig tau pom tias cov khoom sib xyaw ua ke NT thiab fusion proteins muab cov lus qhia siab tshaj tawm thiab muaj txiaj ntsig zoo hydrogels nrog cov khoom zoo nkauj xws li pob tshab, gelation yam tsis muaj kev sib txuas, thiab ncaj qha immobilization ntawm cov protein ntau ntawm qhov ntom ntom.
Kab laug sab muaj ntau li xya kab sib txawv ntawm cov qog, txhua tus tsim ib hom kab lo lo ntxhuav.Tag nrho xya hom kab lo lo ntxhuav yog tsim los ntawm kab laug sab kab laug sab kab laug sab (kab laug sab) kwv yees li 6000 qhov seem ntev thiab muaj thaj tsam loj hauv nruab nrab uas nyob ib puag ncig los ntawm spherical N- thiab C-terminal domains (NT thiab CT) 1,2.Qhov kev kawm dav tshaj plaws ntawm cov kab lo lo ntxhuav, thawj ampulla, yog tsim los ntawm thawj lub caj pas ampulla.Nyob rau hauv lub caj pas no, ib tug monolayer ntawm epithelial hlwb synthesizes spidroin proteins thiab secretes lawv mus rau hauv lub lumen ntawm lub caj pas, qhov chaw uas lawv muaj nyob rau hauv ib tug soluble daim ntawv (doping) ntawm tsis tshua muaj siab concentrations (30-50% w / v) 3,4.Lub koom haum thiab conformation ntawm lub ntsiab ampullar spidroin proteins nyob rau hauv lub caj pas tau debated, tab sis feem ntau cov pov thawj kev sim qhia tau hais tias muaj ib tug feem ntau helical thiab / los yog random helical conformation thiab micellar los yog lamellar qauv 5,6,7,8,9,10.Thaum lub sij hawm rov ua dua tswj hwm cov khoom siv ntawm cov kab lo lo ntxhuav, tsim β-daim ntawv nanocrystals thiab amorphous cov qauv 11,12,13,14,15, kawg tswj tswj cov kab lo lo ntxhuav hauv cov lus teb rau cov kev hloov pauv ntawm cov kab lo lo ntxhuav16,17,18.Los ntawm kev tswj cov kab mob, 19. Terminal domains yog evolutionarily kev txuag thiab lawv cov dej num tej zaum yuav tshwm sim rau tag nrho cov spidroin proteins 2,20,21.Thaum nkag mus rau hauv lub caj pas, pH ntawm spidroin txo los ntawm kwv yees li 7.6 mus rau <5.716 thiab nce nrog shear thiab stretch mediated los ntawm kev txav mus los ntawm qhov maj mam nqaim duct.Hauv kev daws teeb meem, CT yog ib qho α-helical constitutive parallel dimer17, tab sis nyob rau hauv cov lus teb rau pH qis thiab shear rog, CT nthuav tawm thiab hloov β-layers16, 17, tejzaum nws ua rau β-txheej nyob rau hauv cov cheeb tsam rov ua dua ntawm Hloov 16. NT yog monomeric hauv qab. Cov xwm txheej uas cuam tshuam txog cov xwm txheej hauv lub lumen ntawm lub caj pas thiab kho cov solubility ntawm spidroin, tab sis thaum txo pH, protonation ntawm ib tug xov tooj ntawm carboxylic acid sab chains ua rau dimerization ntawm NT nrog ib tug pKa ntawm kwv yees li 6.5, yog li stabilizing NT thiab kho spidroin loj. qhov ntau.networks 16, 18.Yog li, NT ua lub luag haujlwm tseem ceeb hauv kev tsim filament, hloov los ntawm monomer hauv txheej rau ib qho dimer hauv fiber ntau 23,24,25.NT tseem soluble thiab helical nyob rau hauv tag nrho cov xwm txheej kawm txog hnub tim 16, 18, 19, 20, 26, 27, 28, 29, uas tau tshoov siab nws txoj kev loj hlob raws li daim ntawv lo rau kev tsim cov protein ntau heterologous.
Recombinant mini kab laug sab silk protein, suav nrog ib qho NT, ib cheeb tsam luv luv, ib qho CT, thiab His6 tag (His-NT2RepCT) rau kev ua kom huv, yog li soluble hauv aqueous tsis raws li haiv neeg kab laug sab silk protein thiab ua raws li cov yam ntxwv tseem ceeb ntawm kab laug sab. .25.31 Nws.Nws-NT2RepCT tuaj yeem hloov mus rau hauv cov fibers txuas ntxiv siv lub tshuab biomimetic uas pH 8 soluble txheej yog extruded rau hauv pH 525,32,33,34,35 dej da dej.Bioreactor fermentation ntawm E. coli qhia Nws-NT2RepCT thiab tom qab kev kho mob ua rau > 14 g / L yield tom qab purification.Qhov siab yield, siab solubility, thiab txaus cov lus teb ntawm His-NT2RepCT rau acidic tej yam kev mob yog tag nrho vim hais tias ntawm NT23, 25, 34.
Ntawm no peb qhia txog kev tsim sai ntawm cov pob tshab hydrogels los ntawm cov proteins spidroin recombinant, suav nrog NT ib leeg, los ntawm incubating cov kua protein ntawm 37 ° C.Siv thioflavin T fluorescence (ThT), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR) thiab kis electron microscopy (TEM), peb pom tias NT thiab microspider proteins tau hloov pauv cov qauv rau hauv β-cov ntawv thiab amyloid-zoo li fibrils. thaum gels tsim.Tsis tas li ntawd, fusion proteins ntawm NT thiab ntsuab fluorescent protein (GFP) los yog purine nucleoside phosphorylase (PNP) tsim hydrogels nrog tag nrho cov fusion fragments.High-throughput qhia nyob rau hauv heterologous hosts, ua ke nrog ceev ceev ntawm hydrogels nyob rau hauv lub cev tej yam kev mob, opens muaj peev xwm ntawm cov nqi-zoo ntawm hydrogels nrog engineered functions.
Tsis zoo li feem ntau tshaj tawm cov kab mob spidroin proteins36, Nws-NT2RepCT ruaj khov hauv Tris-HCl tsis nyob ntawm pH 8 thiab tuaj yeem ua kom muaj zog txog 500 mg / mL yam tsis muaj nag lossis daus 25.Yog li ntawd, peb xav tsis thoob thaum pom tias cov protein no ua kom pom tseeb, txhawb nqa tus kheej hydrogels thaum incubated ntawm 37 ° C (Fig. 1b-d).Cov kev tshawb fawb ntxiv tau pom tias Nws-NT2RepCT gelation tau tshwm sim ntau qhov ntau ntawm cov protein ntau (10–300 mg / mL) thiab tias qhov concentration no cuam tshuam nrog lub sijhawm gelation (Fig. 1c thiab Supplementary Fig. 1).Txhawm rau kom paub seb qhov twg ntawm Nws-NT2RepCT kho kom haum hydrogel tsim, peb mam li tshuaj xyuas txhua qhov chaw ntawm tus kheej thiab hauv ntau qhov sib xyaw ua ke siv qhov ntsuas qhov ntsuas hluav taws xob (Daim duab 1a, b).Txhua qhov kev sim cov feem ntawm recombinant spidroin tsim gels (ntawm cov protein ntau ntawm 300 mg / mL) hauv tsawg dua 1 h, tshwj tsis yog rau 2Rep precipitated (Fig. 1b).Qhov no qhia tias NT thiab CT ib leeg, ua ke, lossis cuam tshuam nrog kev rov ua dua, tuaj yeem gel ntawm 37 ° C thiab tias His6 tag tsis cuam tshuam rau cov txheej txheem no rau ib qho tseem ceeb.Muab qhov kev xav zoo uas NT yog cov protein ntau soluble thiab ruaj khov, thiab cov ntawv tshaj tawm yav dhau los ntawm cov spidroin hydrogels recombinant tau txais cov teebmeem gelation rau kev hloov pauv hauv cheeb tsam rov qab thiab / lossis CTs, NT nws tus kheej tuaj yeem.Qhov kev tshawb pom ntawm gelation yog npaj txhij.Ntxiv Table 1) 37, 38, 39. Zoo kawg, NT twb gelled nyob rau hauv 10 feeb ntawm ib tug concentration ntawm ≥ 300 mg/mL (Fig. 1c).Vial inversion thwmsim nrog ntau qhov ntau ntawm NT pom tau tias ntawm> 50 mg / mL cov tshuaj NT gelled sai dua Nws-NT2RepCT ntawm qhov sib thooj (w / v, daim duab 1c).
Schematic sawv cev ntawm ntau yam spidroin tsim tau kawm hauv txoj haujlwm no.b Gel lub sij hawm ntawm 37 ° C rau ntau yam recombinant spidroin proteins (300 mg / mL) txheeb xyuas los ntawm inverting lub vial.CT gel tam sim ntawd yam tsis muaj incubation (<300 mg / mL), 2Rep precipitates (300 mg / mL, 5 hli nplai).c Gel lub sij hawm ntawm His-NT2RepCT thiab NT ntawm qhia cov protein ntau ntawm 37 ° C.d Cov duab ntawm Nws-NT2RepCT thiab NT hydrogels nrog kab laug sab thiab tsab ntawv "NT" luam tawm hauv qab, raws li (ob leeg 200 mg / mL, scale bar 5 mm).
Hydrogels tsim los ntawm ntau yam recombinant spidroin proteins muaj xim sib txawv me ntsis, thiab qhov muag liab qab kev soj ntsuam qhia pom qhov sib txawv ntawm cov pob tshab (Fig. 1b).NT gels yog qhov tseeb tshwj xeeb thaum lwm cov gels ua opaque.Nws-NT2RepCT thiab NT gels muab pov rau hauv cov raj cylindrical tuaj yeem raug tshem tawm ntawm cov pwm tsis zoo (Fig. 1d).
Txhawm rau kuaj seb puas muaj kab laug sab kab laug sab lo lo ntxhuav txheej gel nyob rau hauv cov xwm txheej tam sim no pom tias ua rau gelation ntawm cov kab laug sab spider recombinant, coatings tau sau los ntawm lub caj pas loj ampulla ntawm Swedish choj kab laug sab (Larinioides sclopetarius).Cov txheej txheem tau khaws cia hauv 20 mM Tris-HCl tsis nyob ntawm 50 mg / mL (raws li ntsuas qhov hnyav qhuav), tab sis tsis muaj gelation tau pom thaum lub sij hawm 21 hnub incubation ntawm 37 ° C (Cov duab ntxiv 2a).
Txhawm rau kom suav cov gels no, kev ntsuas rheological tuaj yeem siv los kawm txog cov txheej txheem gelation thiab txiav txim siab tag nrho cov khoom siv kho tshuab.Tshwj xeeb, kev saib xyuas cov qauv khaws cia (elasticity) ntawm qhov kub siab tuaj yeem muab cov ntaub ntawv ntawm cov gelling kub thiab cov khoom viscoelastic ntawm txheej.Kev ntsuas kub nce siab (siv 1 ° C / min ntawm 25-45 ° C, raws li cov kev tshawb fawb yav dhau los uas siv cov khoom lag luam ntuj cov ntsiab lus) 40,41 tau pom tias cov moduli cia ntawm His-NT2RepCT thiab NT cov kev daws teeb meem tau nce nrog qhov kub thiab txias.tau nce ntxiv (Daim duab 2 thiab ntxiv daim duab 3).Qhov tseem ceeb, NT module pib loj hlob ntawm qhov kub qis dua piv rau Nws-NT2RepCT, zoo ib yam nrog lub sijhawm gel sai tau pom thaum NT tau ncaj qha nrog His-NT2RepCT ntawm 37 ° C (Daim duab 1).Tom qab qhov kev poob qis hauv qhov kub thiab txias tom qab, qhov kev cia khoom modulus tsis rov qab los rau qis dua qhov tseem ceeb thiab tseem nyob saum qhov poob modulus (saib Daim duab ntxiv 3), qhia tias thermally irreversible gelation ruaj khov.Tom qab gelation, qhov kawg elastic modulus ranged ntawm 15 mus rau 330 kPa rau His-NT2RepCT hydrogels ntawm ib tug concentration ntawm 100–500 mg / mL, thiab lub kawg elastic modulus rau NT hydrogels (100–500 mg / mL) ranged ntawm 2 mus rau 1400 kPa (Fig. , 2 thiab cov ntaub ntawv ramp tiav) saib Daim duab ntxiv 3).
Kev hloov pauv ntawm qhov kub thiab txias thaum ntsuas ntawm His-NT2RepCT (300 mg / mL) thiab b NT (300 mg / mL) nrog kev co.Cov xib xub qhia txog qhov kub thiab txias, thiab qhov sib dua ntawm cov ntaub ntawv khaws cia ntawm cov ntaub ntawv qhia txog qhov ntsuas ntawm qhov ntsuas torque qis rau cov cuab yeej tshaj li teev los ntawm cov chaw tsim khoom, uas yog qhov ua rau lub suab nrov.c Qhov kawg-module tsub zuj zuj ntawm His-NT2RepCT thiab NT tom qab siab kub (100, 300, thiab 500 mg / mL).Tag nrho cov kev nyeem ntawv module raug coj los ntawm qhov zaus ntawm 0.1 Hz.
Raws li ib txoj hauv kev los tshawb xyuas cov kev hloov pauv uas cuam tshuam nrog gelation, peb tau sau FTIR spectra ntawm Nws-NT2RepCT thiab NT ua ntej thiab tom qab gelation ntawm 37 ° C (Daim duab 3a, b).Raws li kev cia siab, qhov spectra ntawm Nws-NT2RepCT thiab NT cov kev daws teeb meem sib raug rau cov proteins uas qhia α-helix / random coil thib ob qauv, nrog lub suab nrov ntawm 1645 cm-1.Rau ob qho tib si hydrogels, gelation ua rau tsim ntawm ob txhais caj npab nyob rau hauv nruab nrab I band ntawm txog 1617 cm-1 thiab 1695 cm-1 (Fig. 3a, b), qhia txog kev tsim ntawm antiparallel β-daim ntawv qauv.Cov kev hloov pauv no kuj tuaj yeem pom meej nyob rau hauv qhov thib ob derivative thiab qhov sib txawv gelation spectra (Supplementary Fig. 4b).Ob pawg ntawm NT β-txheej tau tshaj tawm ntau dua li ntawm Nws-NT2RepCT, qhia tias tag nrho cov ntsiab lus ntawm β-txheej bands hauv NT hydrogel yog siab dua li ntawm NT2RepCT hydrogel.
FTIR absorption spectra ntawm His-NT2RepCT thiab b NT (ob leeg 500 mg / mL) ua ntej (kev daws teeb meem) thiab tom qab (gel) incubation ntawm 37 ° C.c TEM cov duab ntawm resuspended 50 mg/ml NT2RepCT gels thiab d NT.Scale bar 200nm.e Fiber diameters ntawm His-NT2RepCT thiab NT hydrogels.n = 100 ntsuas fibrils, p <0.0001.Cov kab yuam kev qhia qhov sib txawv ntawm tus qauv.Qhov nruab nrab ntawm qhov yuam kev tuav yog qhov nruab nrab.Ib qho kev sib tw t-kev sib tw (ob-tailed) tau siv rau kev txheeb xyuas kev txheeb xyuas.f ThT fluorescence ntawm ntau yam recombinant spidroin proteins (100 mg / mL) ntawm 37 ° C yam tsis tau co.g NT (100 mg / mL) inoculation sim los ntawm 100 mg / mL NT gel nrog 0%, 5%, 10%, thiab 20% noob.
Kev soj ntsuam ntawm cov gel siv kis tau tus mob electron microscopy (TEM) tau pom tias cov hydrogel muaj xws li amyloid fibrils (Fig. 3c, 3d).NT-formed fibrils tau elongated (5–12 nm nyob rau hauv txoj kab uas hla) thiab unbranched, thaum His-NT2RepCT fibrils tau luv luv nyob rau hauv ntev thiab ho wider nyob rau hauv txoj kab uas hla (7–16 nm) (Fig. 3e).Cov txiaj ntsig no tau tso cai rau peb ua raws li kinetics ntawm fibrosis siv thioflavin T (ThT) assay.Rau tag nrho cov recombinant spidroin proteins, lub teeb liab fluorescent nce thaum kuaj tau incubated ntawm 37 ° C (Fig. 3f, Ntxiv Fig. 5a).Raws li qhov kev tshawb pom no, kev soj ntsuam microscopic ntawm NT thiab His-NT2RepCT nyob rau hauv cov xwm txheej gelling tau nthuav tawm ib qho kev nce hauv ThT fluorescence yam tsis muaj kev pom hauv zos ntawm ThT-positive aggregates (Ntxiv Fig. 5b,c).Kev tsim ntawm ThT-zoo fibrils tsis tau nrog kev nce hauv NT thiab Nws-NTCT turbidity (Supplementary Fig. 5d), uas txhais tau hais tias lub network ntawm fibrils hauv gel tuaj yeem tsim yam tsis muaj kev cuam tshuam gel clarity.Kev cog qoob loo los ntawm kev ntxiv me me ntawm pre-formed fibrils tuaj yeem ua kom cov fibril tsim ntawm qee cov amyloids42,43,44 tab sis ntxiv 5%, 10% lossis 20% (w / w) NT rau cov tshuaj NT hydrocoagulants.noob nyhuv (Fig. 3g).Tej zaum qhov no yog vim lub fact tias cov fibrils nyob rau hauv lub hydrogel yog kuj ruaj thiab siv tsis tau raws li cov noob.
Tus cwj pwm tsis txaus ntseeg ntawm cov protein sib xyaw ua ke ntawm qhov kub thiab txias tau ua rau muaj kev tshawb fawb ntxiv ntawm nuclear magnetic resonance (NMR) spectroscopy los txheeb xyuas cov kev hloov pauv cuam tshuam nrog kev tsim gel.NMR spectra ntawm Nws-NT2RepCT cov kev daws teeb meem tau sau tseg nyob rau lub sijhawm ntawm 37 ° C pom tias CT tseem yog ib feem ntawm cov ntaub qhwv, qhov NT thiab 2Rep cov cim tau ploj mus (Daim duab 4a), qhia tias nws yog NT thiab 2Rep uas tswj qee qhov tsim ntawm Nws- NT2RepCT hydrogel.Lub teeb liab CT kuj tau txo qis rau 20% ntawm nws qhov kev siv thawj zaug, qhia tias CT kuj feem ntau ruaj thiab koom ua ke rau hauv cov qauv hydrogel.Rau ib feem me me ntawm CT, uas yog mobile li nyob rau hauv cov qauv preincubated thiab yog li pom los ntawm kev daws NMR, lub spectra tsis muaj cov cim qhia rau thawj 10 cov khoom seem, tejzaum nws yog vim nyuaj immobilization ntawm qhov txuas ntawm Nws-NT2Rep.Lub NMR spectra ntawm -state ntawm hydrogels -NT2RepCT qhia qhov tseem ceeb ntawm α-helices thiab β-txheej txheem thiab, rau ib tug tsawg dua, qhov random coil conformation (Fig. 4b).Kev tshuaj xyuas kev hloov pauv tshuaj ntawm methionine residues tam sim no tsuas yog hauv NT pom tau tias qhov chaw no tau hloov mus rau β-daim qauv.Lub sij hawm-dependent spectra ntawm NT nyob rau hauv kev daws pom ib tug uniform txo nyob rau hauv lub teeb liab siv (Fig. 4c), thiab solid-state NMR ntawm NT hydrogels qhia tau hais tias feem ntau ntawm cov NT residues tau hloov dua siab tshiab rau β-daim ntawv lug (Fig. 4d).Qhov kev sib haum xeeb ntawm 2Rep tsis tuaj yeem txiav txim siab cais vim nws nyiam sib sau ua ke.Txawm li cas los xij, cov khoom hauv lub xeev NMR spectra ntawm NTCT thiab His-NT2RepCT hydrogels zoo sib xws (Fig. 4b; Ntxiv Fig. 6b), qhia tias 2Rep pab txhawb me ntsis rau cov qauv ntawm His-NT2RepCT hydrogel.Rau CT hydrogels, α-helices, β-phau ntawv, thiab random helical theem nrab cov qauv tau pom muaj nyob (Ntxiv Fig. 6d).Qhov no qhia tias qee qhov ntawm CT nyob twj ywm α-helices thaum lwm tus ua β-cov ntawv.Yog li, cov txiaj ntsig ntawm NMR spectroscopy qhia tias NT yog qhov tseem ceeb rau kev tsim hydrogel thiab tseem hloov mus rau hauv β-daim ntawv conformation thaum fusion nrog 2Rep thiab CT.Raws li qhov no, peb nyuam qhuav pom tias amyloid spatial zippers yuav tsim nyob rau hauv tag nrho tsib lub helices ntawm NT sau, thiab Waltz algorithm kwv yees thaj tsam amyloidogenic hauv helix 1 (Fig. 4e).
2D spectra ntawm 15N-HSQC 10 mg/mL His-NT2RepCT tov ua ntej (xiav) thiab 19 teev tom qab incubation (liab) ntawm 37 ° C.Tus neeg hla peaks nyob rau hauv liab spectrum thiab F24, G136, polyA nyob rau hauv lub xiav spectrum yog denoted los ntawm ib tsab ntawv amino acid cim thiab seem seem.Cov insets qhia qhov dependence ntawm lub teeb liab siv lub sij hawm rau cov residues xaiv los ntawm NT, 2Rep, thiab CT domains.b Solid-state radiofrequency (RFDR) spectra ntawm His-NT2RepCT hydrogels.Kev sib raug zoo ntawm Cα / Cβ residues pom hauv RFDR spectra tau txiav txim siab los ntawm kev sib piv nrog cov qauv peptide tshuaj hloov pauv thiab cov txiaj ntsig tau los ntawm kev txheeb cais 82,83 thiab lawv cov qauv theem nrab.SSB – rotating sideband.c Ib-dimensional spectra ntawm 15N-HSQC 10 mg/mL NT tov thaum incubation ntawm 37 ° C rau 36 teev.Lub inset qhia tau hais tias volumetric siv piv rau lub sij hawm.d Khoom lub xeev RFDR spectra ntawm NT hydrogels.Cov kev sib raug zoo ntawm Cα / Cβ residues thiab lawv cov qauv theem ob pom hauv RFDR spectra tau qhia.e Raws li NT45.79 fibrillation propensity profile los ntawm Zipper database (https://services.mbi.ucla.edu/zipperdb/).Lub zog Rosetta ntawm lub spatial xob laim hloov qhov rais ntawm hexapeptide yog qhia hauv kcal / mol.Cov kab liab qhia txog hexapeptides nrog lub siab fibrosis propensity (Rosetta zog hauv qab -23 kcal / mol; hauv qab kab dotted).Cov kab ntsuab qhia cov khoom tawg nrog Rosetta energies saum qhov chaw pib thiab yog li ntawd tsis tshua muaj cov zippers steric.Fragments uas muaj proline raug tshem tawm los ntawm kev tsom xam (tsis muaj kab).Squares qhia thaj chaw ntawm amyloidosis kwv yees los ntawm Waltz algorithm81 (https://waltz.switchlab.org).Qhov sib lawv liag ntawm cov amino acid residues ntawm NT yog nyob rau saum toj, thiab cov hom residues pom nyob rau hauv β theem nrab qauv (txiav txim siab los ntawm solid-state NMR spectroscopy) yog qhia nyob rau hauv liab.Cov haujlwm ntawm tsib NT α-helices raug xaiv raws li (H1-H5)28.
Ntawm pH <6.5, HT dimerizes, tiv taus tshav kub-los yog urea-induced denaturation18.Txhawm rau kom meej meej li cas NT dimerization thiab kev ruaj ntseg cuam tshuam rau gelation, cov kev daws teeb meem uas muaj 100 mg / ml NT tau tswj hwm ntawm pH 8, 7, thiab 6 siv cov vial inversion test.NT cov qauv incubated ntawm pH 8 thiab 7 gelled tom qab 30 min ntawm 37 ° C, tab sis cov pH 8 gel tseem pom tseeb, thaum lub pH 7 gel pom ib qho dej nag (Daim duab 5a).Hauv qhov sib piv, cov tshuaj uas muaj HT ntawm pH 6 tsis tsim cov gel, thiab cov nag lossis daus loj tuaj yeem pom tom qab 20 min ntawm 37 ° C.Qhov no qhia tias dimers lawv tus kheej thiab / lossis lawv qhov kev ruaj ntseg dua piv rau monomers tiv thaiv gelation.Kev tsim cov precipitate rau NT ntawm pH 7 thiab 6 tsis xav tau, txij li nws tau tshaj tawm tias NT yog soluble ntawm 200 mg / ml27, yooj yim refolds tom qab tshav kub denaturation, thiab tseem khaws ib qho α-helix ntawm qis tus nqi ntawm pH 18. Ib qho kev piav qhia txog qhov tsis sib xws yog tias qhov kev sim tau tshaj tawm yav dhau los tau ua nyob rau hauv chav tsev kub lossis qis dua, lossis ntawm cov protein tsawg tsawg 16,18,19.
NT vial inversion test (100 mg/mL) ntawm pH 8, 7, 6 thiab 154 mM NaCl (pH 8) tom qab incubation ntawm 37 ° C.b NT CD spectra nrog thiab tsis muaj 154 mM NaF thiab 154 mM NaCl, feem.Molar ellipticity ntawm 222 nm yog hloov mus rau ib feem ntawm ntuj folds.c NT inversion assay (100 mg / mL) NT * (37 ° C thiab 60 ° C), NTA72R (37 ° C), thiab His-NT-L6 (37 ° C thiab 60 ° C).d CD spectra of NT mutants NT*, NTA72R, and His-NT-L6.Molar ellipticity ntawm 222 nm yog hloov mus rau ib feem ntawm ntuj folds.e Inversion test of NTFlSp, NTMiSp thiab txo NTMiSp (100 mg/mL).Scale bar 5 mm.f CD spectra of NT, NTFlSp, NTMiSp thiab txo NTMiSp.Molar ellipticity ntawm 222 nm yog hloov mus rau ib feem ntawm ntuj folds.Tag Nrho NT spectra ntawm 25 ° C thiab 95 ° C yog qhia nyob rau hauv Ntxiv Daim duab 8.
Physiological ntsev concentration txiav txim siab electrostatic cuam tshuam ntawm NT subunits thiab dimerization ntawm NT hloov mus rau qis pH18.Peb pom tias muaj 154 mM NaCl thiab NaF muaj tseeb inhibit gelation, raws li (Fig. 5a, b; Supplementary Fig. 2b) thiab hais tias cov ntsev no nce lub thermal stability ntawm NT monomers (Fig. 5b, Ntxiv Fig. 8) .Nws kuj qhia tias kev txhim kho kev ruaj ntseg, tsis yog dimerization, tiv thaiv gel tsim.
Txhawm rau tshawb nrhiav ntxiv lub luag haujlwm ntawm cov protein dimerization thiab kev ruaj ntseg hauv gelation, peb siv ob lub mutants, NT * thiab NTA72R, uas tseem nyob twj ywm monomeric ntawm qis pH28.30.NT * yog ib qho kev hloov pauv ob npaug uas qhov pom tseeb dipolar tus nqi faib ntawm cov monomer yog flattened, uas tiv thaiv dimerization thiab ua rau muaj kev ruaj ntseg monomer.NTA72R yog tus nqi dipole, tab sis Arg-hloov Ala nyob ntawm thaj tsam dimer, yog li kev hloov pauv cuam tshuam nrog subunit kev sib cuam tshuam uas yuav tsum tau muaj rau dimerization.Thaum incubation ntawm 37 ° C, NT * tsis tsim hydrogel, thaum NTA72R tsim opaque gel rau 15 min (Fig. 5c).Txij li thaum ob qho tib si NT * thiab NTA72R tsis tuaj yeem dimerize tab sis txawv ntawm monomer stability (Fig. 5d), cov txiaj ntsig tau pom zoo tias qhov kub siab thermal stability tiv thaiv NT los ntawm gelling.Qhov no kuj tau txais kev txhawb nqa los ntawm qhov tseeb tias HT * tsim cov gel thaum nws tsis ruaj khov ntawm qhov kub thiab txias (tom qab 8 min ntawm 60 ° C; Fig. 5c).Nws tau dhau los ua pov thawj tias cov ntsiab lus siab ntawm methionine hauv NT liquefies nws cov folding ntuj thiab qhov 6 Met rau Leu hloov pauv (hu rau ntawm no yog His-NT-L6) ua kom ruaj khov rau NT46 monomer.Raws li qhov kev xav tias kev hloov pauv ntawm cov qauv tsim nyog rau NT gel tsim, peb pom tias His-NT-L6 ruaj khov mutant tsis gel ntawm 37 ° C (Daim duab 5c, d).Txawm li cas los xij, Nws-NT-L6 kuj tsim ib lub gel thaum incubation ntawm 60 ° C rau 60 min (Fig. 5c).
Lub peev xwm ntawm NT hloov mus rau hauv β-daim ntawv cov qauv thiab daim ntawv hydrogels zoo nkaus li siv rau qee qhov tab sis tsis yog tag nrho cov NT domains ntawm spidroin.NTs los ntawm ntau hom kab laug sab thiab kab laug sab, Trichonephila clavipes (NTFlSp), tsim cov gels txawm tias lawv cov ntsiab lus methionine qis thiab siab thermal stability (Fig. 5e, f thiab Supplementary Table 2).Nyob rau hauv sib piv, NT los ntawm me me ampullar protein spidroin los ntawm Araneus ventricosus (NTMiSp) uas tsis tshua muaj thermal stability thiab siab methionine cov ntsiab lus tsis tsim hydrogels (Cov lus ntxiv 2 thiab daim duab 5e, f).Cov tom kawg tuaj yeem cuam tshuam nrog kev muaj cov tshuaj intramolecular disulfide bonds29,47.Tsis tas li ntawd, thaum lub disulfide bonds ntawm NTMiSp raug txo, nws tsim ib hydrogel tom qab incubation ntawm 37 ° C rau 10 min (Fig. 5e).Hauv kev xaus, nws yuav tsum tau muab sau tseg tias cov qauv kev hloov pauv yog ib qho tseem ceeb, tab sis tsis yog tib qho, cov txheej txheem rau kev tsim cov gel los ntawm NT.Lwm qhov tseem ceeb uas yuav muaj feem cuam tshuam yog qhov kev xav tau los tsim cov amyloid fibrils, thiab kev tsom xam nrog zipper database thiab Waltz algorithm tau qhia txog kev sib raug zoo ntawm lub peev xwm tsim cov gels thiab muaj cov cheeb tsam amyloidogenic, nrog rau thaj tsam ntawm thaj tsam kwv yees. tsim steric zippers.Muaj kev sib raug zoo (Table 2 thiab Supplementary Fig. 9).
Lub peev xwm ntawm NT los tsim cov fibrils thiab daim ntawv gels nyob rau hauv cov xwm txheej zoo coj peb mus rau kev xav tias NT fusions nrog lwm cov protein fragments tseem tuaj yeem tsim cov gels nrog kev ua haujlwm tag nrho ntawm cov neeg koom nrog fusion.Txhawm rau kuaj qhov no, peb tau qhia txog cov protein fluorescent ntsuab (GFP) thiab purine nucleoside phosphorylase (PNP) ntawm C-terminus ntawm NT, feem.Qhov tshwm sim fusion proteins tau nthuav tawm hauv E. coli nrog cov txiaj ntsig siab kawg (150 mg / L thiab 256 mg / L co cov kab lis kev cai rau His-NT-GFP thiab His-NT-PNP, raws li), raws li qhov tau pom. rau Lwm cov proteins fused rau NT Ref.30. Nws-NT-GFP (300mg/mL) thiab His-NT-PNP (100mg/mL) fusion proteins tsim gels tom qab 2 teev thiab 6.5 teev ntawm 37 ° C thiab, qhov tseem ceeb, GFP feem tseem tsis hloov.pom tom qab gelation, nrog> 70% ntawm qhov pib fluorescence siv ntxiv tom qab gelation (Fig. 6a).Txhawm rau ntsuas PNP kev ua haujlwm hauv nws-NT-PNP cov kev daws teeb meem thiab cov gels, peb yuav tsum tau dilute cov protein fusion nrog NT vim tias cov kev ua haujlwm ntawm cov enzymatic ntawm kev npaj ntshiab yog sab nraum qhov kev tshawb nrhiav ntau yam ntawm kev soj ntsuam ntawm gelling concentrations.Cov gel tsim nrog cov sib tov uas muaj 0.01 mg / mL His-NT-PNP thiab 100 mg / mL NT khaws cia 65% ntawm qhov pib ua haujlwm enzymatic ntawm cov qauv preincubated (Fig. 6b).Lub gel tseem nyob ruaj khov thaum ntsuas (Ntxiv daim duab 10).
Ib tug txheeb ze fluorescence siv ua ntej thiab tom qab gelation ntawm His-NT-GFP (300 mg / mL) thiab inverted vial muaj His-NT-GFP hydrogel (300 mg / mL) nyob rau hauv pom thiab UV teeb.Cov ntsiab lus qhia cov kev ntsuas tus kheej (n = 3), qhov yuam kev bar qhia tus qauv sib txawv.Qhov nruab nrab tus nqi yog qhia nyob rau hauv nruab nrab ntawm qhov yuam kev tuav.b PNP kev ua haujlwm tau txais los ntawm kev tshuaj xyuas fluorometric siv cov kev daws teeb meem thiab cov gels uas muaj NT (100 mg / ml) thiab cov sib xyaw uas muaj 0.01 mg / ml nws-NT-PNP thiab 100 mg / ml New Taiwan duas.Lub inset qhia ib lub vial inverted uas muaj hydrogel uas muaj His-NT-PNP (5 hli scale bar).
Ntawm no, peb qhia txog kev tsim cov hydrogels los ntawm NT thiab lwm cov recombinant spidroin proteins los ntawm incubating ib tug protein tov ntawm 37 ° C (Daim duab 1).Peb pom tias gelation yog txuam nrog kev hloov pauv ntawm α-helices rau hauv β-txheej txheem thiab tsim ntawm amyloid-zoo li fibrils (Fig. 3 thiab 4).Qhov kev tshawb pom no yog qhov xav tsis thoob vim tias NTs yog coiled globular tsib-helix bundles paub txog lawv cov solubility siab heev thiab ruaj khov siab ntawm cov concentrations> 200 mg / mL ntawm 4 ° C rau ob peb hnub27.Tsis tas li ntawd, NTs tuaj yeem rov ua dua tom qab cua sov denaturation ntawm cov protein tsawg hauv µM.Raws li peb cov txiaj ntsig, kev tsim fibril yuav tsum muaj kev sib xyaw ua ke ntawm> 10 mg / mL protein concentration thiab kub siab me ntsis (Daim duab 1).Qhov no yog ua raws li lub tswv yim hais tias amyloid fibrils tuaj yeem tsim los ntawm globularly folded proteins uas nyob rau hauv ib feem ntawm lub xeev unfolded vim thermal fluctuations nyob rau hauv physiological tej yam kev mob 48 .Piv txwv ntawm cov proteins uas tau txais kev hloov pauv no suav nrog insulin49,50, β2-microglobulin, transthyretin thiab lysozyme51,52,53.Txawm hais tias NT yog ib qho α-helix nyob rau hauv nws haiv neeg lub xeev, kwv yees li 65% ntawm cov polypeptide saw yog sib xws nrog steric zipper tsim (Fig. 4e) 45 .Txij li thaum lub monomer yog dynamically mobile46, nws tuaj yeem nthuav tawm cov cheeb tsam amyloidogenic muaj peev xwm ntawm qhov ntsuas kub nruab nrab thiab ntawm qhov siab ntawm tag nrho cov protein tuaj yeem ncav cuag qhov tseem ceeb rau amyloid fibril formation54.Ua raws li qhov laj thawj no, peb pom qhov tsis zoo ntawm kev sib raug zoo ntawm spidroin concentration thiab gelation lub sij hawm (Daim duab 1c), thiab yog tias cov monomeric NT conformation yog ruaj khov los ntawm kev hloov pauv (NT *, His-NT-L6) los yog ntsev ntxiv, tuaj yeem tiv thaiv cov tsim hydrogels (Fig. 5).
Feem ntau, amyloid fibrils ploj los ntawm kev daws raws li precipitate, tab sis nyob rau hauv qee yam mob lawv tuaj yeem tsim hydrogels55,56,57.Hydrogel-forming fibrils feem ntau muaj qhov sib piv siab thiab tsim cov kev sib txuas ntawm peb-seem ruaj khov los ntawm molecular entanglement, 55,58 raws li peb cov txiaj ntsig.Rau kev tsim hydrogel hauv vitro, cov proteins feem ntau yog tag nrho lossis ib nrab nthuav dav, piv txwv li, los ntawm kev raug cov kuab tshuaj organic, kub siab (70-90 ° C) thiab / lossis pH qis (1.5-3.0) 59,60,61,62.Spidroin hydrogels tau piav qhia ntawm no tsis tas yuav ua haujlwm hnyav, thiab tsis xav tau cov neeg sawv cev sib txuas kom ruaj khov rau cov hydrogels.
Nws tau raug tshaj tawm yav dhau los tias spidroin rov ua dua thiab QDs, uas tshwm sim rau kev hloov pauv β-daim ntawv thaum lub sij hawm txhob lo lo ntxhuav, tsim hydrogels.Piv nrog rau peb qhov kev tshawb pom, lub sij hawm incubation thiab / los yog incubation kub tau ntev dua los yog siab dua, feem, thiab qhov tshwm sim hydrogels feem ntau opaque (Daim duab 7 thiab ntxiv Table 1) 37, 38, 63, 64, 65, 66, 67, 68 , 69. Ntxiv rau lub sij hawm ceev ceev, NT hydrogels > 300 mg/mL (30%) outperformed tag nrho lwm cov recombinant kab laug sab silk protein hydrogels, nrog rau tej yam ntuj tso hydrogels xws li gelatin, alginate (2%), agar (0.5%). ) thiab collagen.(0.6%) (Daim duab 7 thiab Cov Lus Qhia Ntxiv 1 thiab 3) 37,39,66,67,68,69,70,71,72,73,74.
Lub sij hawm gel thiab elastic modulus ntawm hydrogels nyob rau hauv txoj kev tshawb no tau muab piv nrog rau lwm cov spidroin-based hydrogels thiab xaiv natural hydrogels.Cov ntaub ntawv raug muab nrog rau cov lus piav qhia ntawm cov txheej txheem gelation.APS Ammonium persulfate, chav tsev kub.Cov ntaub ntawv 37, 38, 39, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
Kab laug sab zoo li tau tsim txoj hauv kev los tiv thaiv kab laug sab los ntawm gelling thaum khaws cia.Txawm hais tias muaj qhov siab ntawm cov protein ntau hauv cov qog nqaij hlav, qhov loj rov ua dua ntawm thaj chaw txuas nrog lub davhlau ya nyob twg txhais tau hais tias qhov pom tseeb ntawm NT thiab CT hauv caj pas sib raug rau kwv yees li 10-20 mg / ml, ntawm thaj tsam ntawm txoj kev tshawb no.xav tau rau hauv vitro pom hydrogel tsim.Tsis tas li ntawd, cov ntsiab lus zoo sib xws ntawm cov ntsev 16 stabilized NT, xws li hauv cov qog nqaij hlav (Fig. 5b).NT conformation tau kawm nyob rau hauv E. coli cytosol thiab pom tias yuav nruj nreem folded tshaj thaum kuaj nyob rau hauv vitro, ntxiv qhia tias ntsev los yog lwm yam tseem ceeb tiv thaiv nws aggregation nyob rau hauv vivo.Txawm li cas los xij, lub peev xwm ntawm NTs los hloov mus rau hauv β-daim ntawv fibrils tej zaum yuav yog ib qho tseem ceeb rau kev tsim filament thiab yuav tsum tau tshawb xyuas hauv kev tshawb fawb yav tom ntej.
Ntxiv rau qhov tshiab ntawm NT-amyloid-zoo li fibril thiab hydrogel tsim tau pom nyob rau hauv txoj kev tshawb no, peb kuj qhia tau tias qhov tshwm sim no yuav muaj kev siv biotechnological thiab biomedical (Fig. 8).Raws li cov ntaub ntawv pov thawj ntawm lub tswv yim, peb ua ke NT nrog GFP lossis PNP thiab pom tias cov fusion protein kuj tsim hydrogels thaum incubated ntawm 37 ° C thiab tias GFP thiab PNP feem ntau khaws lawv cov dej num tom qab gelation (Daim duab 6).Nucleoside phosphorylase yog ib qho tseem ceeb catalysts synthesis ntawm nucleoside analogues75, uas ua rau peb nrhiav tau cuam tshuam rau kev lag luam biopharmaceutical.Lub tswv yim ntawm kev nthuav tawm fusion proteins uas tsim cov pob tshab hydrogels nyob rau hauv cov kev mob zoo tso cai rau kev tsim cov hydrogels functionalized nrog cov khoom zoo rau ntau yam kev siv xws li enzyme immobilization, tswj cov tshuaj tso tawm thiab cov ntaub so ntswg engineering.Tsis tas li ntawd, NT thiab NT * yog cov cim qhia tau zoo 30, uas txhais tau hais tias NT thiab nws cov kev hloov pauv tuaj yeem siv rau kev tsim cov fusion ntawm cov proteins uas muaj kuab heev thiab tom qab tsim cov immobilized lub hom phiaj proteins hauv 3D hydrogels.
NT yog soluble, α-helical thiab ruaj khov ntawm qis concentrations (µM) thiab 37 ° C.Ntawm qhov kub thiab txias, tab sis ntawm qhov nce siab (> 10 mg / ml), NT cov ntaub ntawv gels uas muaj amyloid-zoo li fibrils.NT fusion proteins kuj tsim fibrillar gels nrog kev ua haujlwm zoo fusion fragments, tso cai rau ntau yam proteins kom immobilized hauv 3D hydrogels siv NT.Hauv qab: NT (PDB: 4FBS) thiab cov duab piav qhia ntawm fiber ntau tes hauj lwm thiab cov qauv protein txuam (xws li thiab tsis kos rau qhov ntsuas, GFP PDB: 2B3Q, 10.2210/pdb2B3Q/pdb; PNP PDB: 4RJ2, 10.2210/pdb4).
Cov kev tsim kho (saib Daim Ntawv Ntxiv 4 rau ib daim ntawv teev tag nrho suav nrog cov amino acid sequences) tau cloned rau hauv plasmid pT7 thiab hloov mus rau E. coli BL21 (DE3).E. coli uas muaj cov plasmids engineered tau inoculated hauv Luria broth ntxiv nrog kanamycin (70 mg / l) thiab loj hlob ib hmos ntawm 30 ° C thiab 250 rpm.Cov kab lis kev cai ces inoculated 1/100 rau hauv LB nruab nrab uas muaj kanamycin thiab kab lis kev cai ntawm 30 ° C thiab 110 rpm kom txog thaum OD600 mus txog 0.8.Rau cov kev tshawb fawb NMR, cov kab mob tau loj hlob hauv M9 qhov nruab nrab tsawg uas muaj 2 g ntawm D-glucose 13C (Aldrich) thiab 1 g ntawm ammonium chloride 15N (Cambridge Isotope Laboratories, Inc.) rau kev sau cov protein nrog isotopes.Txo qhov kub thiab txias mus rau 20 degrees Celsius thiab induce protein qhia nrog 0.15 mM isopropylthiogalactopyranoside (kawg concentration).Tom qab hmo ntuj qhia cov protein, cov hlwb tau sau ntawm 7278 × g, 4 ° C rau 20 min.Cell pellets raug tshem tawm hauv 20 mM Tris-HCl, pH 8, thiab khov kom txog thaum siv ntxiv.Thawed hlwb tau lysed siv lub cell disruptor (TS series machines, Constant Systems Limited, England) ntawm 30 kPa.Tom qab ntawd cov lysates tau centrifuged ntawm 25,000 g rau 30 feeb ntawm 4 ° C.Rau NTMiSp, cov pellet tau rov qab muab tso rau hauv 2 M urea, 20 mM Tris-HCl, pH 8, thiab sonicated rau 2 min (2 s rau / tawm, 65%), ces centrifuged dua ntawm 25,000 xg, 4 ° C. 30 feeb.Lub supernatant tau thauj mus rau Ni-NTA kem, ntxuav nrog 20 mM Tris-HCl, 2 mM imidazole, pH 8, thiab thaum kawg cov protein tau eluted nrog 20 mM Tris-HCl, 200 mM imidazole, pH 8. Los tsim NT2RepCT thiab NTCT, thrombin digestion qhia qhov chaw (ThrCleav) ntawm Nws thiab NT.Thrombin cleavage sites kuj muaj nyob rau hauv His-NT-ThrCleav-2Rep (produces 2Rep), His-thioredoxin-ThrCleav-NT (produces NT), His-thioredoxin-ThrCleav-CT (produces CT), His-Thioredoxin-ThrCleav-NT .* (produces NT*), His-Thioredoxin-ThrCleav-NTA72R (produces NTA72R), His-Thioredoxin-ThrCleav-NTFlSp (produces NTF1Sp), and His-Sulphur Redoxin-ThrCleav-NTMiSp (produces NTMiSp).Cov khoom tsim tau zom nrog thrombin (1:1000) thiab dialyzed ib hmos ntawm 4 ° C. nrog 20 mM Tris-HCl, pH 8, siv Spectra / Por dialysis membrane nrog ib qho hnyav molecular pib ntawm 6-8 kDa.Tom qab kev lim ntshav, cov kev daws teeb meem yog thauj mus rau Ni-NTA kem thiab cov effluent uas muaj cov protein uas txaus siab tau sau.Protein concentrations tau txiav txim siab los ntawm kev ntsuas UV absorbance ntawm 280 nm siv cov extinction coefficient ntawm txhua cov protein, tshwj tsis yog rau NTF1Sp, uas siv Bradford assay raws li cov chaw tsim khoom raws tu qauv.Purity yog txiav txim los ntawm SDS polyacrylamide (4-20%) gel electrophoresis thiab Coomassie ci ntsa iab xiav staining.Proteins tau concentrated siv centrifuge lim (VivaSpin 20, GE Healthcare) ntawm 4000 xg nrog 10 kDa molecular hnyav txiav tawm hauv 20 feeb mus.
Thaw cov tshuaj protein thiab ua tib zoo pipet 150 µl rau hauv 1 ml ntshiab septum vial (8 x 40 mm Thermo Scientific).Cov raj tau capped thiab kaw nrog parafilm kom tiv thaiv evaporation.Cov qauv (n = 3) tau muab tso rau ntawm 37 ° C lossis 60 ° C thiab ua ntu zus los saib xyuas gelation.Cov qauv uas tsis gel tau incubated tsawg kawg yog ib lub lim tiam.Txo NTMiSp disulfide bonds nrog 10 mM DTT ib 10 µM protein.Txhawm rau txheeb xyuas cov gelation ntawm cov kab laug sab kab laum ntuj, Swedish choj kab laug sab raug txiav, ob lub qog loj ampullated tau muab tso rau hauv 200 μl ntawm 20 mM Tris-HCl tsis pH 8 thiab txiav kom tso cai rau cov txheej txheem cais tawm ntawm cov qog..Cov ntsiab lus ntawm cov qog yog yaj nyob rau hauv tsis, 50 µl rau kev txiav txim siab qhuav qhov hnyav (los ntawm incubation ntawm qhib vials ntawm 60 ° C rau qhov hnyav) thiab 150 μl rau gelation ntawm 37 ° C.
Kev ntsuas geometry / cov cuab yeej yog ua los ntawm cov hlau tsis muaj steel siv cov phaj sib txuas nrog rau sab saum toj txoj kab uas hla ntawm 20 hli thiab qhov sib txawv ntawm 0.5 hli.Ua kom sov cov qauv ntawm 25 ° C mus rau 45 ° C thiab rov qab mus rau 25 ° C ntawm tus nqi ntawm 1 ° C ib feeb twg siv lub stainless hlau hauv qab Peltier phaj.Kev ntsuas vibrational tau ua nyob rau ntawm qhov zaus ntawm 0.1 Hz thiab nyob rau hauv cheeb tsam linear viscoelastic ntawm cov khoom ntawm ib hom ntawm 5% thiab 0.5% rau cov qauv ntawm 100 mg / mL thiab 300-500 mg / mL, feem.Siv lub chamber kev cai los tiv thaiv evaporation.Cov ntaub ntawv tau txheeb xyuas siv Prism 9.
Rau sau infrared (IR) spectra ntawm chav tsev kub ntawm 800 txog 3900 cm-1.Cov cuab yeej ATR, nrog rau txoj kev teeb pom kev los ntawm lub spectrometer, yog purged nrog qhuav lim cua ua ntej thiab thaum lub sij hawm sim.Cov kev daws teeb meem (500 mg / mL kom txo qis qhov nqus dej hauv qhov ntsuas) tau muab tso rau hauv cov muaju, thiab cov gels (500 mg / mL) tau tsim ua ntej ntsuas thiab tom qab ntawd hloov mus rau cov muaju (n = 3).1000 scans tau kaw nrog kev daws teeb meem ntawm 2 cm-1 thiab xoom lub voj voog ntawm 2. Qhov thib ob derivative tau suav nrog OPUS (Bruker) siv qhov sib luag ntawm cuaj lub ntsiab lus.Cov spectra tau normalized rau tib cheeb tsam kev koom ua ke ntawm 1720 thiab 1580 cm-1 siv F. Menges "Spectragryph - Optical Spectroscopy Software".Nyob rau hauv ATR-IR spectroscopy, qhov tob tob ntawm ib tug infrared beam mus rau hauv ib tug qauv yog wavenumber nyob ntawm seb, ua rau muaj zog nqus nyob rau hauv qis wavenumbers ntau dua wavenumbers.Cov teebmeem no tsis tau raug kho rau qhov spectra qhia hauv Fig.3 vim lawv muaj me me heev (Ntxiv daim duab 4).Kho qhov spectra rau daim duab no tau suav nrog Bruker OPUS software.
Nyob rau hauv txoj cai, ib qho kev nthuav dav ntawm cov protein conformations yog ua tau tom qab txhim khu kev qha deconvolution ntawm cov khoom nyob rau hauv lub amide kuv ncov.Txawm li cas los xij, qee qhov teeb meem tshwm sim hauv kev xyaum.Lub suab nrov hauv lub spectrum tuaj yeem tshwm sim li (tsis tseeb) peaks thaum lub sij hawm deconvolution.Tsis tas li ntawd, lub ncov vim dej khoov coincides nrog txoj hauj lwm ntawm amide kuv ncov thiab tej zaum yuav muaj ib tug zoo sib xws rau cov qauv uas muaj ib tug loj npaum li cas ntawm cov dej, xws li cov aqueous gel kawm ntawm no.Yog li ntawd, peb tsis tau sim kom decompose tag nrho cov amide kuv ncov, thiab peb cov kev soj ntsuam yuav tsum tsuas yog xav nyob rau hauv kev txhawb nqa ntawm lwm txoj kev xws li NMR spectroscopy.
Cov kev daws teeb meem ntawm 50 mg / ml NT thiab His-NT2RepCT tau gelled hmo ntuj ntawm 37 ° C.Tom qab ntawd cov hydrogel tau diluted nrog 20 mM Tris-HCl (pH 8) mus rau ib qho concentration ntawm 12.5 mg / ml, shaken zoo thiab pipetted kom tawg cov gel.Tom ntej no, hydrogel tau diluted 10 zaug nrog 20 mM Tris-HCl (pH 8), 5 μl ntawm cov qauv yog siv rau ib daim phiaj tooj liab coated nrog formvar, thiab cov qauv dhau los raug tshem tawm nrog daim ntawv blotting.Cov qauv raug ntxuav ob zaug nrog 5 µl ntawm MilliQ dej thiab stained nrog 1% uranyl formate rau 5 feeb.Tshem tawm cov stain ntau dhau nrog cov ntawv nqus dej, tom qab ntawd cua qhuav lub mesh.Kev yees duab tau ua tiav ntawm cov phiajcim no siv FEI Tecnai 12 Ntsuj Plig BioTWIN ua haujlwm ntawm 100 kV.Cov duab tau kaw ntawm x 26,500 thiab x 43,000 magnifications siv Veleta 2k × 2k CCD lub koob yees duab (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).Rau txhua tus qauv (n = 1), 10–15 cov duab raug kaw.ImageJ (https://imagej.nih.gov/) tau siv rau kev tsom xam cov duab thiab ntsuas ntawm fiber ntau diameters (n = 100, fibers sib txawv).Prism 9 tau siv los ua qhov kev xeem tsis sib xws (ob-tailed).Qhov txhais tau tias Nws-NT2RepCT thiab NT fibrils yog 11.43 (SD 2.035) thiab 7.67 (SD 1.389) nm, feem.Lub sijhawm cog qoob loo (95%) yog -4.246 txog -3.275.qib kev ywj pheej = 198, p <0.0001.
80 µl ntawm cov qauv ua kua uas muaj 10 µM thioflavin T (ThT) tau ntsuas hauv triplicate (n = 3) nyob rau hauv cov xwm txheej zoo li qub siv Corning 96-zoo dub hauv qab daim hlau hauv qab (Corning Glass 3881, USA).Fluorescence sib txawv tau sau tseg siv 440 nm excitation lim thiab 480 nm emission lim (FLUOStar Galaxy los ntawm BMG Labtech, Offenburg, Lub teb chaws Yelemees).Lub teeb liab ThT tsis tau saturated los yog quenched, raws li kev sim nrog ntau ntau ntawm ThT tau ua tsis tau hloov lub teeb liab siv.Sau ntawv absorbance ntawm 360 nm rau kev ntsuas huab cua.Rau kev sim cov noob, 100 mg / mL gels tau tsim ntawm 37 ° C., resuspended, thiab siv rau cov noob ntawm molar ratios ntawm 5%, 10%, thiab 20%.Cov ntaub ntawv tau txheeb xyuas siv Prism 9.
Thaw cov khoom ntawm His-NT2RepCT thiab NT> 100 mg / mL rau dej khov thiab lim los ntawm 0.22 µm lim.Concentrations raug xam los ntawm kev ntsuas absorbance ntawm 280 nm siv Nanodrop.Hauv qhov dej ntawm 96-zoo dub tsis khi phaj (Corning) nrog lub hauv qab ntshiab, cov qauv tau diluted rau 20 mg / ml hauv 20 mM Tris-HCl pH 8 thiab tov nrog 5 μM ThT (qhov kawg concentration), tag nrho cov qauv concentration 50ml ua.Cov qauv tau muab tso rau txhua 10 feeb ntawm 37 ° C ntawm CellObserver (Zeiss) microscope nrog kis lub teeb channel thiab FITC excitation thiab emission lim teeb rau ThT duab.Lub lens 20x / 0.4 yog siv los ua duab.Zen Blue (Zeiss) thiab ImageJ (https://imagej.nih.gov/) tau siv rau kev tsom xam duab.Gels kuj tau npaj los ntawm NT thiab His-NT2RepCT cov kev daws teeb meem ntawm qhov concentration ntawm 50 mg / mL uas muaj 20 mM Tris pH 8 thiab 5 µM ThT thiab incubated ntawm 37 ° C rau 90 min.Cov gel daim tau raug xa mus rau lub qhov dej tshiab uas muaj 20 mM Tris, pH 8, thiab 5 μM ThT nyob rau hauv ib qho tsis khi dub 96 zoo meej hauv qab phaj.Tau txais ntsuab fluorescence thiab cov duab ci ntsa iab ntawm 20x / 0.4 magnification.ImageJ tau siv los txheeb xyuas cov duab.
Kev daws NMR spectra tau txais ntawm 310 K ntawm 600 MHz Bruker Avance Neo spectrometer nruab nrog QCI Quadrupole Resonance Pulsed Gradient Field Cryoprobe (HFCN).NMR cov qauv uas muaj 10 mg/mL homogeneous protein sau nrog 13C, 15N, yaj hauv 20 mM Tris-HCl (pH 8), 0.02% (w/v) NaN3, 5% DO (v/v), (n = 1) .Kev hloov tshuaj lom neeg ntawm NT2RepCT ntawm pH 6.7 tau siv los muab qhov siab tshaj 23 hauv 2D spectrum ntawm 15N-HSQC.
Lub kaum ntse ntse spinning khoom NMR (MAS) spectra ntawm 13C, 15N-labeled hydrogels tau kaw rau ntawm Bruker Avance III HD spectrometer ntawm 800 MHz nruab nrog 3.2 hli 13C / 15N{1H} electronless sojntsuam.Cov qauv ntsuas kub tau tswj tau siv qhov ntsuas kub tsis sib xws ntawm 277 K. Ob-dimensional dipole rotational resonance (DARR)76 thiab xov tooj cua zaus reconnection (RFDR)77 spectra tau txais ntawm MAS zaus ntawm 12.5 kHz thiab 20 kHz, feem.Hla polarization (CP) los ntawm 1H mus rau 13C yog ua los ntawm linear ramp ntawm 60.0 mus rau 48.0 kHz ntawm 1H, 61.3/71.6 kHz ntawm 13C (ntawm 12.5/20 kHz MAS) thiab lub sij hawm tiv tauj 0.5–1 ms.Spinal6478 decoupling ntawm 73.5 kHz tau siv thaum sau cov ntaub ntawv.Lub sijhawm tau txais yog 10 milliseconds thiab lub voj voog qeeb yog 2.5 vib nas this.Ib qho kev sib txuas ntawm Cα / Cβ kev sib raug zoo tau pom hauv RFDR spectra tau muab los ntawm cov yam ntxwv ntawm cov tshuaj residue-hom tshuaj hloov pauv thiab kev sib txuas sib txuas hauv DARR spectra.
Lub Zipper79 database (https://services.mbi.ucla.edu/zipperdb/) tau siv los soj ntsuam flutter tendencies thiab Rosetta zog rau NT, NTFlSp, thiab NTMiSp.Lub Zipper database suav Rosetta Energy80, uas muab ntau lub zog pub dawb rau cov qauv thiab txheeb xyuas cov qauv protein.Lub zog theem ntawm -23 kcal / mol lossis qis dua qhia tias muaj kev nyiam ua rau fibrillate.Lub zog qis txhais tau tias muaj kev ruaj ntseg ntau dua ntawm ob lub β-strands hauv zipper conformation.Tsis tas li ntawd, Waltz algorithm tau siv los kwv yees thaj tsam amyloidogenic hauv NT, NTFlSp thiab NTMiSp Ref.81. (https://waltz.switchlab.org/).
Cov tshuaj NT protein yog tov nrog 2-(N-morpholino) ethanesulfonic acid (MES) tsis nyob ntawm pH 5.5 thiab 6.0 kom txo cov pH mus rau pH 6 thiab 7, feem.Qhov kawg ntawm cov protein ntau yog 100 mg / ml.
Kev ntsuas tau ua tiav ntawm J-1500 CD spectrometer (JASCO, USA) siv 300 μL cuvette nrog txoj kev kho qhov muag ntawm 0.1 cm.Proteins tau diluted rau 10 μM (n = 1) hauv 20 mM phosphate buffer (pH 8).Txhawm rau txheeb xyuas cov protein ruaj khov nyob rau hauv lub xub ntiag ntawm ntsev, cov proteins tau txheeb xyuas ntawm tib qhov concentration (n = 1) hauv 20 mM phosphate buffer (pH 8) uas muaj 154 mM NaF lossis NaCl, feem.Kev ntsuas kub tau sau tseg ntawm 222 nm ntawm 25 ° C txog 95 ° C nrog cua sov ntawm 1 ° C / min.Qhov kev faib ua feem ntawm ib txwm folded proteins yog xam los ntawm cov mis (KDmeasure – KDfinal)/(KDstart – KDfinal).Tsis tas li ntawd, tsib spectra tau sau tseg rau txhua tus qauv ntawm 260 nm txog 190 nm ntawm 25 ° C thiab tom qab cua sov mus rau 95 ° C.Tsib spectra tau nruab nrab, smoothed thiab hloov mus rau molar ellipticity.Cov ntaub ntawv tau txheeb xyuas siv Prism 9.
Qhov kev siv fluorescence ntawm His-NT-GFP (300 mg / mL, 80 µL) tau ntsuas hauv triplicate (n = 3) hauv 96-zoo Corning daim hlau nrog cov pob tshab dub hauv qab (Corning Glass 3881, USA) nyob rau hauv cov xwm txheej zoo li qub.Ntsuas cov qauv nrog lub fluorescence-raws li phaj nyeem ntawv nrog ib qho excitation wavelength ntawm 395 nm thiab sau cov emission ntawm 509 nm ua ntej gelation thiab 2 teev tom qab ntawm 37 ° C.Cov ntaub ntawv tau txheeb xyuas nrog Prism 9.
Purine nucleoside phosphorylase kev ua haujlwm kuaj cov khoom siv (fluorometric method, Sigma Aldrich) tau siv raws li cov chaw tsim khoom cov lus qhia.Txhawm rau ntsuas kev ua haujlwm hauv cov gels thiab cov kev daws teeb meem uas muaj His-NT-PNP, sib tov 10 ng ntawm Nws-NT-PNP nrog 100 mg / mL NT rau tag nrho cov ntim ntawm 2 µL vim tias cov gel tau muab lub teeb liab saum toj ntawm lub sijhawm tshawb pom ntawm lub teeb.Kev tswj xyuas cov gels thiab cov kev daws teeb meem uas tsis muaj His-NT-PNP suav nrog.Kev ntsuas tau ua ob zaug (n = 2).Tom qab kev ua haujlwm tau ntsuas, cov tshuaj tiv thaiv sib xyaw raug tshem tawm thiab cov gel tau yees duab los xyuas kom meej tias cov gel tseem nyob hauv lub sijhawm ntsuas.Cov ntaub ntawv tau txheeb xyuas siv Prism 9.
Yog xav paub ntxiv txog kev tsim qauv, saib Nature study abstract linked to this article.
Daim duab 1 thiab 2 nthuav qhia thawj cov ntaub ntawv.1c, 2a–c, 3a, b, e–g, 4, 5b, d, f, thiab 6, Supplementary Fig.3, ntxiv fig.5a ,d ,qw.6 thiab ntxiv daim duab.8. Cov ntaub ntawv cov ntaub ntawv los ntawm txoj kev tshawb no yog tuav hauv Zenodo database https://doi.org/10.5281/zenodo.6683653.Cov ntaub ntawv NMR tau txais hauv qhov kev tshawb fawb no tau muab tso rau hauv BMRBig chaw cia khoom hauv qab nkag ID bmrbig36.Cov qauv ntawm GFP thiab PNP raug coj los ntawm PDB (GFP 2B3Q, PNP 4RJ2).
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