310 10 * 1mm Chitsulo chosapanga dzimbiri chophimbidwa ndi mankhwala, Magawo a N-terminal a spidroin amapanga ma hydrogel otengera ma amyloid fibrils ndikupereka nsanja yolepheretsa mapuloteni.

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Kufotokozera

310 10 * 1mm Chitsulo chosapanga dzimbiri chophimbidwa ndi ogulitsa machubu

Chemical Composition

Mechanical Properties

Mapuloteni a kangaude ophatikizana (mapuloteni a silika a kangaude) ali ndi ntchito zambiri zomwe zingagwiritsidwe ntchito popanga ma biomaterials atsopano, koma mawonekedwe awo ophatikizika komanso ophatikizika amawapangitsa kukhala ovuta kuwapeza komanso osavuta kugwiritsa ntchito.Apa tikunena kuti mapuloteni ang'onoang'ono a spidroin ndipo, chofunikira kwambiri, dera la N-terminal (NT) lokha limapanga ma hydrogel odzithandizira okha komanso owonekera pa 37 ° C.Mapuloteni ophatikizika okhala ndi NT ndi mapuloteni obiriwira a fulorosenti kapena purine nucleoside phosphorylase amapanga mapuloteni osakanikirana bwino.Hydrogels.Zotsatira zathu zikuwonetsa kuti mapuloteni ophatikizana a NT ndi ma fusion amapereka zokolola zapamwamba kwambiri ndipo amapatsa ma hydrogel okhala ndi zinthu zowoneka bwino monga kuwonekera, gelation popanda kuphatikizika, komanso kusasunthika mwachindunji kwa mapuloteni omwe amagwira ntchito pakuchulukira kwakukulu.
Akangaude ali ndi tiziwalo 7 tosiyanasiyana ta silika, ndipo iliyonse imatulutsa mtundu winawake wa silika.Mitundu yonse isanu ndi iwiri ya silika imapangidwa ndi ma spider silk proteins (spidroins) pafupifupi 6000 otsalira ndipo ali ndi gawo lalikulu lapakati lobwereza lozunguliridwa ndi madera ozungulira a N- ndi C-terminal (NT ndi CT)1,2.Mtundu wophunziridwa kwambiri wa silika, woyamba ampula, umapangidwa ndi khwangwala loyamba la ampula.Mu gland iyi, monolayer wa maselo a epithelial amapanga mapuloteni a spidroin ndikuwabisa mu lumen ya gland, kumene amakhala mu mawonekedwe osungunuka (doping) pamagulu okwera kwambiri (30-50% w / v)3,4.Kukonzekera ndi kugwirizana kwa mapuloteni akuluakulu a ampullar spidroin mu gland akhala akukangana, koma umboni wambiri woyesera umasonyeza kukhalapo kwa mawonekedwe a helical ndi / kapena osasinthika ndi mapangidwe a micellar kapena lamellar5,6,7,8,9,10.Ngakhale madera obwerezabwereza amayang'anira makina a ulusi wa silika, kupanga β-sheet nanocrystals ndi mapangidwe amorphous11,12,13,14,15, madera otsiriza amayang'anira ulusi wa silika potengera kusintha kwa zinthu pamodzi ndi silika gland16,17,18.Poyang'anira mapangidwe a silika, 19. Magawo apakati amasungidwa mwachisinthiko ndipo ntchito yawo ikhoza kukhala yofanana ndi mapuloteni onse a spidroin 2,20,21.Pakudutsa mu gland, pH ya spidroin imatsika kuchoka pa 7.6 kufika ku <5.716 ndipo imawonjezeka ndi kumeta ubweya ndi kutambasula molumikizana ndi kuyenda kudzera mu njira yochepetsetsa pang'onopang'ono.Mu yankho, CT ndi α-helical constitutive parallel dimer17, koma poyankha pH yochepa ndi mphamvu zometa ubweya, CT imatsegula ndi kusintha β-layers16, 17, mwinamwake kuyambitsa β-zigawo m'madera obwerezabwereza a Convert 16. NT ndi monomeric pansi pawo. mikhalidwe yowonetsa mikhalidwe mu lumen ya chithokomiro ndikuyimira kusungunuka kwa spidroin, koma pH ikachepa, kupangika kwa maunyolo angapo am'mbali mwa carboxylic acid kumabweretsa kuchepetsedwa kwa NT ndi pKa pafupifupi 6.5, potero kukhazikika kwa NT ndikukonza spidroin yayikulu. kuchuluka.maukonde16,18.Choncho, NT imagwira ntchito yofunika kwambiri pakupanga ulusi, kusintha kuchokera ku monomer mu zokutira kupita ku dimer mu fiber23,24,25.NT imakhalabe yosungunuka kwambiri komanso ya helical pansi pamikhalidwe yonse yomwe idaphunziridwa mpaka pano16, 18, 19, 20, 26, 27, 28, 29, yomwe idalimbikitsa chitukuko chake ngati cholembera chowonjezera cha solubility chopanga mapuloteni a heterologous.
Recombinant mini spider silk protein, yopangidwa ndi NT imodzi, chigawo chachifupi chobwereza, CT imodzi, ndi His6 tag (His-NT2RepCT) kuti iyeretsedwe, imasungunuka m'madzi am'madzi monga mapuloteni a kangaude wamba ndipo amatsanzira zofunikira za kangaude wa silika. .Chithunzi cha 25.31.His-NT2RepCT imatha kupota mu ulusi wosalekeza pogwiritsa ntchito makina a biomimetic momwe zokutira zosungunuka za pH 8 zimatulutsidwa mu bafa lamadzi la pH 525,32,33,34,35.Kuwotchera kwa E. coli kwa bioreactor kusonyeza His-NT2RepCT ndi chithandizo cha pambuyo pake kunapangitsa kuti> 14 g/L zokolola zitayeretsedwa.Zokolola zambiri, kusungunuka kwakukulu, komanso kuyankha kokwanira kwa His-NT2RepCT ku mikhalidwe ya acidic zonse zimatengera NT23, 25, 34.
Apa tikunena za kupangidwa mwachangu kwa ma hydrogel owonekera kuchokera ku mapuloteni ophatikizanso a spidroin, kuphatikiza NT yokha, poyika njira ya protein pa 37 °C.Pogwiritsa ntchito thioflavin T fluorescence (ThT), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR) ndi transmission electron microscopy (TEM), tinapeza kuti NT ndi ma microspider proteins amasinthidwa kukhala ma β-sheets ndi fibrils ngati amyloid. pamene ma gels amapangidwa.Kuphatikiza apo, mapuloteni ophatikizika a NT ndi mapuloteni obiriwira a fulorosenti (GFP) kapena purine nucleoside phosphorylase (PNP) amapanga ma hydrogel okhala ndi tiziduswa tating'onoting'ono tosakanikirana.Kufotokozera kwapamwamba kwambiri mu makamu a heterologous, kuphatikizidwa ndi kupangika mwachangu kwa ma hydrogel pansi pamikhalidwe yakuthupi, kumatsegula mwayi wopanga ma hydrogel otsika mtengo okhala ndi ntchito zopangidwira.
Mosiyana ndi mapuloteni ambiri a spidroin36, His-NT2RepCT imakhala yokhazikika mu Tris-HCl buffer pa pH 8 ndipo imatha kukhazikika mpaka 500 mg/mL popanda mpweya25.Choncho, tinadabwa kupeza kuti puloteniyi imapanga mofulumira ma hydrogel optically, odzithandizira okha pamene amapangidwa ndi 37 ° C (mkuyu 1b-d).Kafukufuku wina anasonyeza kuti His-NT2RepCT gelation zinachitika pa osiyanasiyana mapuloteni ndende (10-300 mg/mL) ndi kuti ndende imeneyi anali inversely correlated ndi gelation nthawi (mkuyu. 1c ndi Supplementary mkuyu. 1).Kuti mudziwe kuti ndi magawo ati a His-NT2RepCT mediate hydrogel mapangidwe, tidayang'ana dera lililonse payekhapayekha komanso mophatikizira zosiyanasiyana pogwiritsa ntchito kuyesa kwa botolo (Chithunzi 1a,b).Zigawo zonse zoyesedwa za recombinant spidroin zinapanga ma gels (pa protein ndende ya 300 mg / mL) pasanathe 1 h, kupatulapo precipitated 2Rep (mkuyu. 1b).Izi zikusonyeza kuti NT ndi CT yokha, kuphatikizapo, kapena yogwirizana ndi kubwerezabwereza, ikhoza kusungunuka pa 37 ° C komanso kuti chizindikiro cha His6 sichimakhudza njirayi pamlingo uliwonse waukulu.Poganizira zomwe anthu ambiri amaganiza kuti NT ndi puloteni yosungunuka kwambiri komanso yokhazikika, komanso kuti malipoti am'mbuyomu a recombinant spidroin hydrogels akuti zotsatira za ma gelation ndi kusintha kosinthika m'magawo obwereza ndi/kapena ma CTs, NT yokha imatha.Kupezeka kwa gelation kunali kosayembekezereka.Supplementary Table 1) 37, 38, 39. Chodabwitsa, NT kale gelled mkati 10 mphindi pa ndende ya ≥ 300 mg/mL (mkuyu. 1c).Kuyesa kwa vial kusinthika kosiyanasiyana kwa NT kunawonetsa kuti> 50 mg/mL njira ya NT idapangidwa mwachangu kuposa His-NT2RepCT pamlingo wofananira (w/v, Chithunzi 1c).
Kuyimira kwadongosolo kwamapangidwe osiyanasiyana a spidroin omwe aphunziridwa mu ntchitoyi.b Nthawi ya gel osakaniza pa 37 °C ya mapuloteni osiyanasiyana a spidroin (300 mg/mL) amatsimikiziridwa ndi kutembenuza vial.CT gel nthawi yomweyo popanda makulitsidwe (<300 mg/mL), 2Rep precipitates (300 mg/mL, 5 mm sikelo).c Nthawi ya Gel ya His-NT2RepCT ndi NT pazambiri zama protein pa 37°C.d Zithunzi za His-NT2RepCT ndi NT hydrogels ndi kangaude ndi chilembo "NT" chosindikizidwa pansi, motsatira (onse 200 mg/mL, sikelo 5 mm).
Ma Hydrogel omwe amapangidwa ndi mapuloteni osiyanasiyana a spidroin amakhala ndi mitundu yosiyana pang'ono, ndipo kuyang'ana maso kumawonetsa kuwonekera mosiyanasiyana (mkuyu. 1b).Ma gelisi a NT amamveka bwino kwambiri pomwe ma gelisi ena amakhala opaque.Ake-NT2RepCT ndi NT angelo kuponyedwa mu cylindrical machubu akhoza kuchotsedwa nkhungu bwinobwino (mkuyu. 1d).
Kuti muwone ngati zokutira za kangaude wachilengedwe zimapaka utoto wa kangaude pansi pamikhalidwe yomwe tsopano yapezeka kuti imapangitsa kuti mapuloteni a spidroin apangidwenso, zokutira zidasonkhanitsidwa kuchokera ku kangaude wamkulu wa Swedish Bridge (Larinioides sclopetarius).Zovalazo zinasungidwa mu 20 mM Tris-HCl buffer pa 50 mg / mL (kutengera kulemera kowuma), koma palibe gelation yomwe inawonedwa panthawi ya 21 incubation pa 37 ° C (Supplementary Figure 2a).
Kuti muwerenge ma gels awa, miyeso ya rheological ingagwiritsidwe ntchito pophunzira momwe ma gelation amagwirira ntchito ndikuzindikira mawonekedwe onse amakina.Makamaka, kuyang'anira modulus yosungiramo (kusungunuka) pa kutentha kwapamwamba kungapereke chidziwitso cha kutentha kwa gelling komanso maonekedwe a viscoelastic a zokutira.Kuyesera kukwera kwa kutentha (pogwiritsa ntchito 1 ° C / min pa 25-45 ° C, kutengera maphunziro apitalo pogwiritsa ntchito njira zachilengedwe za silika) 40,41 inasonyeza kuti moduli yosungiramo njira za His-NT2RepCT ndi NT zinawonjezeka ndi kutentha kwakukulu .chinawonjezeka (mkuyu 2 ndi Supplementary mkuyu. 3).Makamaka, gawo la NT linayamba kukula pa kutentha kochepa poyerekeza ndi His-NT2RepCT, yogwirizana ndi nthawi yofulumira ya gel yomwe inawonedwa pamene NT inalumikizidwa mwachindunji ndi His-NT2RepCT pa 37 ° C (Chithunzi 1).Pambuyo pakutsika kwa kutentha, modulus yosungirayo sinabwerere kuzinthu zotsika ndikukhalabe pamwamba pa modulus yotayika (onani Supplementary Fig. 3), kusonyeza gelation yosasinthika.Pambuyo pa gelation, chomaliza chotanuka modulus kuyambira 15 mpaka 330 kPa kwa His-NT2RepCT hydrogels pa ndende ya 100-500 mg/mL, ndipo chomaliza zotanuka modulus kwa NT hydrogels (100-500 mg/mL) ankachokera 2 mpaka 1400 kPa (Mkuyu, 2 ndi deta yonse ya ramp) onani Supplementary Fig. 3).
a Kusintha kwa kutentha pa miyeso ya His-NT2RepCT (300 mg/mL) ndi b NT (300 mg/mL) ndi kugwedezeka.Mivi imawonetsa kutentha, ndipo mawonekedwe opepuka a gawo losungiramo data akuwonetsa kuyesedwa pamitengo yotsika ya torque ya chida kuposa momwe wopanga adafotokozera, zomwe ndizomwe zimayambitsa phokoso.c Kumapeto kwa gawo la His-NT2RepCT ndi NT pambuyo pa kutentha kwakukulu (100, 300, ndi 500 mg/mL).Zowerengera zonse za module zimatengedwa pafupipafupi 0.1 Hz.
Monga njira yomwe ingatheke kufufuza zosintha zamapangidwe okhudzana ndi gelation, tidajambulitsa mawonekedwe a FTIR a His-NT2RepCT ndi NT isanakwane ndi pambuyo pa 37 ° C (Chithunzi 3a,b).Monga zikuyembekezeredwa, mawonekedwe a His-NT2RepCT ndi NT mayankho amafanana ndi mapuloteni omwe akuwonetsa α-helix/random coil yachiwiri, yokhala ndi gulu lodziwika pa 1645 cm-1.Kwa ma hydrogel onse, gelation inachititsa kuti manja awiri apangidwe pakati pa I band pafupifupi 1617 cm-1 ndi 1695 cm-1 (mkuyu 3a, b), kusonyeza mapangidwe antiparallel β-mapepala.Zosinthazi zitha kuwonekanso momveka bwino mu gawo lachiwiri lomwe limachokera komanso kusiyana kwa gelation spectra (Supplementary Fig. 4b).Magulu awiri a NT β-layer anali omveka kwambiri kuposa a His-NT2RepCT, kusonyeza kuti zonse zomwe zili m'magulu a β-layer mu NT hydrogel zinali zapamwamba kuposa za NT2RepCT hydrogel.
a FTIR mayamwidwe sipekitiramu wa His-NT2RepCT ndi b NT (onse 500 mg/mL) pamaso (yankho) ndi pambuyo (gel osakaniza) incubation pa 37°C.c Zithunzi za TEM zoyimitsidwanso za 50 mg/ml NT2RepCT gels ndi d NT.Scale bar 200 nm.e Fiber diameters of His-NT2RepCT ndi NT hydrogels.n = 100 miyezo ya fibrils, p <0.0001.Zolemba zolakwika zikuwonetsa kupatuka kokhazikika.Pakatikati pa mipiringidzo yolakwika ndiye tanthauzo.Kuyesa kwa t kosasinthika (kwa michira iwiri) kunagwiritsidwa ntchito posanthula ziwerengero.f ThT fluorescence ya mapuloteni osiyanasiyana a spidroin (100 mg/mL) pa 37 °C osagwedezeka.g NT (100 mg/mL) kuyesa katemera kuchokera ku 100 mg/mL NT gel osakaniza ndi 0%, 5%, 10%, ndi 20% mbewu.
Kuwunika kwa gel ogwiritsira ntchito ma electron microscopy (TEM) kunasonyeza kuti hydrogel imakhala ndi ma fibrils amyloid (Fig. 3c, 3d).Mafinya opangidwa ndi NT anali otalikirapo (5-12 nm m'mimba mwake) ndipo alibe nthambi, pamene mafinya Ake-NT2RepCT anali aafupi m'litali komanso okulirapo kwambiri (7-16 nm) (Mkuyu 3e).Zotsatirazi zidatilola kutsatira kinetics ya fibrosis pogwiritsa ntchito mayeso a thioflavin T (ThT).Kwa mapuloteni onse a spidroin, chizindikiro cha fulorosenti chinawonjezeka pamene zitsanzo zinayikidwa pa 37 ° C (mkuyu 3f, Supplementary Fig. 5a).Mogwirizana ndi izi, kuyezetsa kwapang'onopang'ono kwa NT ndi His-NT2RepCT pansi pamikhalidwe ya gelling kunawonetsa kuwonjezeka kofanana kwa ThT fluorescence popanda kudzikundikira komweko kwa magulu a ThT-positive aggregates (Supplementary Fig. 5b, c).Mapangidwe a ThT-positive fibrils sanali limodzi ndi kuwonjezeka kwa NT ndi His-NTCT turbidity (Supplementary Fig. 5d), zomwe zikutanthauza kuti ma network a fibrils mu gel amatha kupanga popanda kusokoneza kumveka kwa gel.Kubzala mbewu powonjezera ulusi wopangidwa kale kumatha kufulumizitsa kwambiri kupanga kwa ma amyloids42,43,44 koma kuwonjezera 5%, 10% kapena 20% (w/w) NT ku yankho la NT hydrocoagulants.seeding zotsatira (mkuyu. 3g).Mwina izi ndichifukwa choti ma fibrils mu hydrogel ndi okhazikika ndipo sangagwiritsidwe ntchito ngati mbewu.
Makhalidwe osayembekezeka a mapuloteni ophatikizananso a spidroin pa kutentha kwakukulu adalimbikitsa maphunziro owonjezera a nyukiliya maginito a resonance (NMR) kuti azindikire kusintha kogwirizana ndi mapangidwe a gel.Mawonekedwe a NMR a mayankho a His-NT2RepCT omwe adalembedwa pakapita nthawi pa 37 ° C adawonetsa kuti CT idapindidwa pang'ono, pomwe ma NT ndi ma sign a 2Rep anali atasowa (mkuyu 4a), kutanthauza kuti makamaka NT ndi 2Rep omwe amawongolera pang'ono mapangidwe Ake- NT2RepCT hydrogel.Chizindikiro cha CT chidatsitsidwanso ku 20% ya mphamvu yake yoyambirira, kutanthauza kuti CT imakhazikikanso ndikuphatikizidwa mu kapangidwe ka hydrogel.Kwa gawo laling'ono la CT, lomwe limakhala loyenda ngati muyeso womwe udayikidwa kale ndipo potero kuwonedwa ndi yankho la NMR, mawonekedwewo alibe zizindikiro zotsalira 10 zoyambira, mwina chifukwa cha kusasunthika kwa gawo lomwe laphatikizidwa la His-NT2Rep.Mawonekedwe a NMR a -state of hydrogels -NT2RepCT adawonetsa kukhalapo kwakukulu kwa α-helices ndi β-layers ndipo, pang'onopang'ono, mawonekedwe a coil mwachisawawa (mkuyu 4b).Kusanthula kwakusintha kwamankhwala kwa zotsalira za methionine zomwe zimapezeka mu NT zokha zidawonetsa kuti domain iyi idasinthidwa kukhala β-sheet.Chiwonetsero chodalira nthawi ya NT mu njira yothetsera vutoli chinasonyeza kuchepa kwa yunifolomu mu mphamvu ya chizindikiro (mkuyu 4c), ndi NMR yolimba ya NT hydrogels inasonyeza kuti zambiri za NT zotsalira zinasinthidwa kukhala β-mapepala (mkuyu 4d).Kugwirizana kwa 2Rep sikungadziwike padera chifukwa cha chizolowezi chake chophatikiza.Komabe, mawonekedwe olimba a NMR a NTCT ndi His-NT2RepCT hydrogels ankawoneka ofanana kwambiri (mkuyu 4b; Supplementary Fig. 6b), kutanthauza kuti 2Rep inathandizira pang'ono ku gawo lachipangidwe cha His-NT2RepCT hydrogel.Kwa CT hydrogels, α-helices, β-mapepala, ndi zowonongeka zowonongeka zowonongeka zinapezeka kuti zilipo (Supplementary Fig. 6d).Izi zikusonyeza kuti mbali zina za CT zimakhalabe α-helices pamene zina zimakhala β-mapepala.Choncho, zotsatira za maonekedwe a NMR zimasonyeza kuti NT ndi yofunika kuti hydrogel ipangidwe komanso imasandulika kukhala β-sheet conformation pa fusion ndi 2Rep ndi CT.Mogwirizana ndi izi, posachedwapa tapeza kuti amyloid spatial zippers mwina amapanga ma helice onse asanu a NT, ndipo ndondomeko ya Waltz inaneneratu za dera la amyloidogenic mu helix 1 (mkuyu 4e).
2D spectra ya 15N-HSQC 10 mg/mL His-NT2RepCT solution isanakwane (buluu) ndi maola 19 pambuyo pa incubation (yofiira) pa 37°C.Payekha mtanda nsonga mu sipekitiramu wofiira ndi F24, G136, polyA mu sipekitiramu buluu amasonyezedwa ndi chilembo chimodzi amino asidi zizindikiro ndi manambala otsalira.Zoyikapo zikuwonetsa kudalira kwamphamvu kwa siginecha pa nthawi ya zotsalira zosankhidwa kuchokera ku madera a NT, 2Rep, ndi CT.b Solid-state radiofrequency (RFDR) mawonekedwe a His-NT2RepCT hydrogels.Kugwirizana kwa zotsalira za Cα / Cβ zomwe zimawonedwa mu RFDR spectra zidatsimikiziridwa poyerekeza ndi ma peptide chemical shift shifts ndi zikhalidwe zochokera ku statistics82,83 ndi zigawo zawo zachiwiri.SSB - gulu lozungulira lozungulira.c One-dimensional sipekitiramu ya 15N-HSQC 10 mg/mL NT yankho pa incubation pa 37 °C kwa 36 hours.Cholembacho chikuwonetsa kuchuluka kwa volumetric motsutsana ndi nthawi.d Solid state RFDR mawonekedwe a NT hydrogels.Kugwirizana kwa zotsalira za Cα / Cβ ndi zigawo zawo zachiwiri zomwe zimawonedwa mu RFDR spectra zikuwonetsedwa.e Kutengera mbiri ya NT45.79 fibrillation propensity kuchokera ku database ya Zipper (https://services.mbi.ucla.edu/zipperdb/).Mphamvu ya Rosetta pawindo losinthira mphezi ya hexapeptide ikuwonetsedwa mu kcal/mol.Mipiringidzo yofiira imatanthawuza ma hexapeptides okhala ndi fibrosis propensity (mphamvu ya Rosetta pansi -23 kcal / mol; pansi pa mzere wa madontho).Mipiringidzo yobiriwira imawonetsa zidutswa zokhala ndi mphamvu za Rosetta pamwamba pa khomo ndipo motero zimakhala zosavuta kupanga zipi zotsekemera.Zidutswa zomwe zili ndi proline sizinaphatikizidwe pakuwunika (popanda mizati).Mabwalo akuwonetsa madera a amyloidosis omwe adanenedweratu ndi Waltz algorithm81 (https://waltz.switchlab.org).Mndandanda wa zotsalira za amino acid za NT zili pamwamba, ndipo mitundu ya zotsalira zomwe zimapezeka mu β yachiwiri (yotsimikiziridwa ndi mawonekedwe olimba a NMR) akuwonetsedwa mofiira.Malo a ma helikisi asanu a NT α amatchulidwa kuti (H1-H5)28.
Pa pH <6.5, HT imachepa, kugonjetsedwa ndi kutentha kapena urea-induced denaturation18.Kuti timvetsetse momwe NT dimerization ndi kukhazikika zimakhudzira gelation, mayankho okhala ndi 100 mg/ml NT amayendetsedwa pa pH 8, 7, ndi 6 pogwiritsa ntchito kuyesa kwa vial inversion.NT zitsanzo incubated pa pH 8 ndi 7 gelled pambuyo 30 min pa 37 ° C, koma pH 8 gel osakaniza anakhalabe bwino, pamene pH 7 gel osakaniza anasonyeza precipitate looneka (mkuyu. 5a).Mosiyana ndi izi, yankho lomwe lili ndi HT pa pH 6 silinapange gel, ndipo mphepo yaikulu imatha kuwoneka pambuyo pa mphindi 20 pa 37 ° C.Izi zikuwonetsa kuti ma dimers okha ndi / kapena kukhazikika kwawo kwakukulu poyerekeza ndi ma monomers amalepheretsa gelation.Kupanga kwa mpweya wa NT pa pH 7 ndi 6 sikunali kuyembekezera, popeza zanenedwa kuti NT imasungunuka pa 200 mg/ml27, imabwereranso mosavuta pambuyo pa kutentha kwa kutentha, komanso imasunga α-helix pamtengo wotsika kwambiri. pH 18. Kufotokozera kothekera kwa kusagwirizanaku ndikuti zoyeserera zomwe zidanenedwapo kale zidachitika kutentha kwachipinda kapena pansi, kapena pama protein otsika kwambiri16,18,19.
NT vial inversion test (100 mg/mL) pa pH 8, 7, 6 ndi 154 mM NaCl (pH 8) pambuyo pa incubation pa 37°C.b NT CD chowonera chokhala ndi 154 mM NaF ndi 154 mM NaCl motsatana.Molar ellipticity pa 222 nm imasinthidwa kukhala gawo la makutu achilengedwe.c NT inversion assay (100 mg/mL) NT* (37 °C ndi 60 °C), NTA72R (37 °C), ndi His-NT-L6 (37 °C ndi 60 °C).d mawonekedwe a CD a NT mutants NT*, NTA72R, ndi His-NT-L6.Molar ellipticity pa 222 nm imasinthidwa kukhala gawo la makutu achilengedwe.e Inversion mayeso a NTFlSp, NTMiSp ndi kuchepetsa NTMiSp (100 mg/mL).Kutalika kwa 5 mm.f ma CD a NT, NTFlSp, NTMiSp komanso kuchepetsedwa kwa NTMiSp.Molar ellipticity pa 222 nm imasinthidwa kukhala gawo la makutu achilengedwe.Mawonekedwe athunthu a NT pa 25 °C ndi 95 °C akuwonetsedwa mu Supplementary Figure 8.
Kuchuluka kwa mchere wamchere kumatsimikizira kuyanjana kwa electrostatic pakati pa ma subunits a NT ndi dimerization ya kusamutsidwa kwa NT kupita kumunsi pH18.Tinapeza kuti kukhalapo kwa 154 mM NaCl ndi NaF kwenikweni kuletsa gelation, motero (mkuyu 5a, b; Supplementary mkuyu. 2b) ndi kuti mchere anawonjezera matenthedwe bata la NT monomers (mkuyu 5b, Supplementary Mkuyu. 8). .Ikuwonetsanso kuti kukulitsa kukhazikika, m'malo mwa dimerization, kumalepheretsa mapangidwe a gel.
Kuti tipitirize kufufuza ntchito ya protein dimerization ndi kukhazikika mu gelation, tinagwiritsa ntchito masinthidwe awiri, NT * ndi NTA72R, omwe amakhalabe monomeric pa pH28.30 yotsika.NT* ndi pawiri mlandu chosinthira kusintha kumene poonekera dipolar mlandu kugawa kwa monoma ndi flattened, amene amalepheretsa dimerization ndi kwambiri kumawonjezera kukhazikika monoma.NTA72R ndi dipole loyipiridwa, koma Arg-substituted Ala ili pamalire a dimer, kotero masinthidwe amasokoneza kuyanjana kwa subunit komwe kumafunikira kuti dimerization.Pamakulitsidwe pa 37 ° C, NT * sinapange hydrogel, pamene NTA72R inapanga gel opaque kwa mphindi 15 (mkuyu 5c).Popeza onse NT * ndi NTA72R sangathe dimerize koma amasiyana monomer bata (mkuyu. 5d), zotsatirazi zikusonyeza kwambiri kuti mkulu thermodynamic bata zimalepheretsa NT ku gelling.Izi zimathandizidwanso ndi mfundo yakuti HT * imapanga gel pamene ili yosakhazikika pa kutentha kwakukulu (pambuyo pa 8 min pa 60 ° C; Chithunzi 5c).Zawonetsedwa kale kuti kuchuluka kwa methionine mu NT kumapangitsa kupukutidwa kwake kwachilengedwe komanso kuti zolowa m'malo zisanu ndi chimodzi za Met to Leu (zotchulidwa pano ngati His-NT-L6) zimakhazikika mwamphamvu NT46 monomer.Kutengera lingaliro lakuti kusinthasintha kwapangidwe kumafunika kuti apange gel osakaniza a NT, tidapeza kuti kusinthika kwa His-NT-L6 sikunapangire gel pa 37 ° C (Chithunzi 5c, d).Komabe, His-NT-L6 inapanganso gel osakaniza pa makulitsidwe pa 60 ° С kwa 60 min (mkuyu 5c).
Kuthekera kwa NT kusandutsa ma β-mapepala ndikupanga ma hydrogel akuwoneka kuti akugwira ntchito kumadera ena koma osati onse a NT a spidroin.Ma NT ochokera kumitundu yosiyanasiyana ya silika ndi akangaude, Trichonephila clavipes (NTFlSp), adapanga ma gels ngakhale kuti ali ndi methionine yochepa komanso kukhazikika kwamafuta ambiri (mkuyu 5e, f ndi Supplementary Table 2).Mosiyana ndi zimenezi, NT kuchokera ku mapuloteni ang'onoang'ono a ampullar spidroin ochokera ku Araneus ventricosus (NTMiSp) omwe ali ndi kutentha kwapakati komanso kutsika kwa methionine sikunapange ma hydrogel (Supplementary Table 2 ndi Fig. 5e, f).Chotsatiracho chikhoza kugwirizanitsidwa ndi kukhalapo kwa intramolecular disulfide bonds29,47.Nthawi zonse, pamene zomangira disulfide NTMiSp anachepetsedwa, izo anapanga hydrogel pambuyo makulitsidwe pa 37 ° C kwa 10 min (mkuyu. 5e).Pomaliza, ziyenera kudziwidwa kuti kusinthasintha kwamapangidwe ndikofunikira, koma osati kokha, mulingo wopangira gel kuchokera ku NT.Chinthu china chomwe chingakhale chofunikira ndi kuthekera kopanga ma amyloid fibrils, komanso kusanthula ndi database ya zipper ndi algorithm ya Waltz kunawonetsa kulumikizana pakati pa kuthekera kopanga ma gels ndi kupezeka kwa zigawo za amyloidogenic, komanso kuchuluka kwa zigawo zomwe zidanenedweratu. kupanga steric zippers.Panali mgwirizano (Table Supplementary Table 2 ndi Supplementary Fig. 9).
Kuthekera kwa NT kupanga ma fibrils ndikupanga ma gels pansi pamikhalidwe yabwino kunatipangitsa kulingalira kuti kuphatikiza kwa NT ndi zidutswa zina zama protein kumatha kupanga ma gels omwe ali ndi ntchito yonse ya ma fusion ogwirizana.Kuti tiyese izi, tinayambitsa mapuloteni obiriwira a fulorosenti (GFP) ndi purine nucleoside phosphorylase (PNP) pa C-terminus ya NT, motero.Mapuloteni osakanikirana adawonetsedwa mu E. coli ndi zokolola zomaliza kwambiri (150 mg/L ndi 256 mg/L shake flask zikhalidwe za His-NT-GFP ndi His-NT-PNP, motsatana), mogwirizana ndi zomwe zawonetsedwa. kwa mapuloteni ena ophatikizidwa ku NT Ref.30. Mapuloteni ake-NT-GFP (300mg / mL) ndi Ake-NT-PNP (100mg / mL) osakanikirana amapanga ma gels pambuyo pa maola a 2 ndi maola 6.5 pa 37 ° C ndipo, chofunika kwambiri, gawo la GFP silinasinthe.anawonedwa pambuyo gelation, ndi> 70% ya chiyambi fulorosisi mphamvu otsala pambuyo gelation (mkuyu. 6a).Kuyeza PNP ntchito yake-NT-PNP njira ndi gels, tinayenera kuchepetsa maphatikizidwe mapuloteni ndi NT chifukwa enzymatic ntchito ya kukonzekera koyera anali kunja kudziwika osiyanasiyana assay pa gelling woipa.The gel osakaniza anapanga ndi osakaniza munali 0,01 mg/mL Ake-NT-PNP ndi 100 mg/mL NT anapitiriza 65% ya koyamba enzymatic ntchito ya preincubated zitsanzo (mkuyu. 6b).Gello linakhalabe losasunthika panthawi ya kuyeza (Supplementary Fig. 10).
Kuchuluka kwa fluorescence isanayambe kapena itatha kutsekemera kwa His-NT-GFP (300 mg/mL) ndi vial inverted yomwe ili ndi His-NT-GFP hydrogel (300 mg/mL) yowoneka ndi kuwala kwa UV.Mfundo zimasonyeza miyeso ya munthu aliyense (n = 3), mipiringidzo yolakwika imawonetsa kupatuka kokhazikika.Mtengo wapakati ukuwonetsedwa pakatikati pa zolakwitsa.b PNP ntchito anapezedwa ndi fluorometric kusanthula ntchito zothetsera ndi gels opangidwa NT (100 mg/ml) ndi osakaniza munali 0.01 mg/ml ake-NT-PNP ndi 100 mg/ml New Taiwan madola.Cholowacho chikuwonetsa mbale yotembenuzidwa yokhala ndi hydrogel yokhala ndi His-NT-PNP (5 mm sikelo bar).
Pano, timapereka lipoti la kupangidwa kwa ma hydrogels kuchokera ku NT ndi mapuloteni ena ophatikizana ndi spidroin mwa kuyika njira ya mapuloteni pa 37 ° C (Chithunzi 1).Timasonyeza kuti gelation imagwirizanitsidwa ndi kusintha kwa α-helices kukhala β-layers ndi kupanga ma fibrils ngati amyloid (Fig. 3 ndi 4).Izi ndizodabwitsa chifukwa NTs ndi globular globular mitolo isanu ya helix yomwe imadziwika kuti imakhala yosungunuka kwambiri komanso yokhazikika pamagulu> 200 mg / mL pa 4 ° C kwa masiku angapo27.Komanso, NTs mosavuta kubwereza pambuyo kutentha denaturation pa otsika mapuloteni woipa mu µM.Malingana ndi zotsatira zathu, mapangidwe a fibril amafuna kuphatikiza> 10 mg / mL mapuloteni osakanikirana ndi kutentha pang'ono (mkuyu 1).Izi zikugwirizana ndi lingaliro lakuti amyloid fibrils akhoza kupanga kuchokera ku mapuloteni opangidwa ndi globularly omwe ali mu gawo losasinthika chifukwa cha kusinthasintha kwa kutentha pansi pa zochitika za thupi 48.Zitsanzo za mapuloteni omwe amatembenuzidwa ndi insulin49,50, β2-microglobulin, transthyretin ndi lysozyme51,52,53.Ngakhale NT ndi α-helix m'dziko lake, pafupifupi 65% ya unyolo wa polypeptide imagwirizana ndi mapangidwe a zipper (mkuyu 4e) 45.Popeza kuti monomer ndi dynamically mobile46, ikhoza kuwonetsa madera omwe angakhale amyloidogenic pa kutentha kokwera kwambiri komanso pamagulu ambiri a mapuloteni amatha kufika pamagulu ovuta kwambiri a amyloid fibril formation54.Kutsatira lingaliro ili, tapeza kulumikizana koyipa pakati pa ndende ya spidroin ndi nthawi ya gelation (mkuyu 1c), ndipo ngati mawonekedwe a monomeric NT akhazikika mwina ndi masinthidwe (NT *, His-NT-L6) kapena kuwonjezera mchere, angalepheretse kupanga ma hydrogels (mkuyu 5).
Nthawi zambiri, amyloid fibrils amatha kuchoka ku yankho ngati mpweya, koma nthawi zina amatha kupanga hydrogels55,56,57.Ma hydrogel-forming fibrils nthawi zambiri amakhala ndi chiyerekezo chokwera kwambiri ndipo amapanga maukonde okhazikika azithunzi zitatu kudzera pama cell entanglement, 55,58 mogwirizana ndi zotsatira zathu.Pakupanga kwa hydrogel mu vitro, mapuloteni nthawi zambiri amawululidwa mokwanira kapena pang'ono, mwachitsanzo, pokhudzana ndi zosungunulira zamoyo, kutentha kwakukulu (70-90 ° C) ndi / kapena pH yochepa (1.5-3.0) 59,60,61,62.Ma spidroin hydrogel omwe akufotokozedwa pano safuna kukonzedwa mwankhanza, komanso safuna othandizira olumikizirana kuti akhazikitse ma hydrogel.
Zanenedwapo kale kuti spidroin imabwereza ndi ma QD, omwe amawoneka kuti akuyenda ndi β-sheet switching panthawi yopota silika, amapanga ma hydrogel.Poyerekeza ndi zomwe tapeza, nthawi zopangira ma incubation ndi / kapena kutentha kwa makulitsidwe kunali kotalikirapo kapena kupitilira apo, motero, ndipo ma hydrogel omwe amakhalapo nthawi zambiri amakhala opaque (Chithunzi 7 ndi Supplementary Table 1) 37, 38, 63, 64, 65, 66, 67, 68 , 69. Kuphatikiza pa nthawi yofulumira ya gel osakaniza, ma hydrogel a NT>300 mg/mL (30%) amaposa ma hydrogel a kangaude a silika, komanso ma hydrogel achilengedwe monga gelatin, alginate (2%), agar (0.5%). ) ndi collagen.(0.6%) (Chithunzi 7 ndi Matebulo Owonjezera 1 ndi 3) 37,39,66,67,68,69,70,71,72,73,74.
Nthawi ya gel ndi zotanuka modulus ya ma hydrogel mu kafukufukuyu adafanizidwa ndi ma hydrogel ena opangidwa ndi spidroin ndi ma hydrogel achilengedwe osankhidwa.Maumboni amaperekedwa limodzi ndi kufotokozera za mikhalidwe ya gelation.APS Ammonium sulfate, kutentha kwa chipinda.Deta 37, 38, 39, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
Akangaude akuwoneka kuti apanga njira zopewera spidroin kuti zisagwe posungira.Ngakhale kuchuluka kwa mapuloteni mu gland ya silika, dera lalikulu lobwerezabwereza lomwe limagwirizanitsidwa ndi malo otsiriza limatanthauza kuti chiwerengero cha NT ndi CT mu gland chimagwirizana ndi pafupifupi 10-20 mg / ml, pamalire a phunziroli.zofunika mu vitro anaona hydrogel mapangidwe.Komanso, ofanana ndende mchere 16 okhazikika NT, monga silika glands (mkuyu. 5b).NT conformation yaphunziridwa mu E. coli cytosol ndipo yapezeka kuti ndi yolimba kwambiri kuposa pamene ikuyang'aniridwa mu vitro, kusonyezanso kuti mchere kapena zinthu zina zimalepheretsa kusonkhanitsa kwake mu vivo.Komabe, kuthekera kwa NTs kusintha kukhala ma β-sheet fibrils kungakhale kofunikira pakupanga ulusi ndipo kuyenera kufufuzidwa m'maphunziro amtsogolo.
Kuphatikiza pa zolemba zatsopano za NT-amyloid-like fibril ndi mapangidwe a hydrogel omwe amawonedwa mu phunziroli, tikuwonetsanso kuti chodabwitsa ichi chikhoza kukhala ndi biotechnological and biomedical applications (Mkuyu 8).Monga umboni wa lingaliro, tinaphatikiza NT ndi GFP kapena PNP ndipo tinasonyeza kuti puloteni yosakanikirana imapanganso ma hydrogel pamene imayikidwa pa 37 ° C komanso kuti magawo a GFP ndi PNP amasunga kwambiri ntchito yawo pambuyo pa gelation (Chithunzi 6).Nucleoside phosphorylases ndizofunikira kwambiri zopangira nucleoside analogues75, zomwe zimapangitsa kuti zomwe tapeza zikhale zogwirizana ndi mafakitale a biopharmaceutical.Lingaliro la kufotokoza mapuloteni ophatikizika omwe amapanga ma hydrogel owonekera pansi pamikhalidwe yabwino amalola kupanga ma hydrogel opangidwa ndi magwiridwe antchito abwino pazinthu zingapo monga enzyme immobilization, kutulutsidwa kwa mankhwala oyendetsedwa ndi umisiri wa minofu.Kuonjezera apo, NT ndi NT* ndi zizindikiro zowonetsera bwino30, zomwe zikutanthauza kuti NT ndi zosiyana zake zitha kugwiritsidwa ntchito popanga mapuloteni osungunuka osungunuka ndi kupanga mapuloteni osasunthika mu 3D hydrogel.
NT imasungunuka, α-helical komanso yokhazikika pazigawo zotsika (µM) ndi 37°C.Pa kutentha komweko, koma pakuchulukirachulukira (> 10 mg/ml), NT imapanga ma gels opangidwa ndi ma fibrils ngati amyloid.Mapuloteni ophatikizika a NT amapanganso ma gels a fibrillar okhala ndi tizidutswa tating'onoting'ono, zomwe zimapangitsa kuti mapuloteni osiyanasiyana asasunthike mu 3D hydrogels pogwiritsa ntchito NT.Pansi: NT (PDB: 4FBS) ndi mafanizo a maukonde a ulusi ndi mapulotini ogwirizana (aganiziridwa komanso osakokedwa pamlingo, GFP PDB: 2B3Q, 10.2210/pdb2B3Q/pdb; PNP PDB: 4RJ2, 10.2210/pdb4RJ2).
Zomanga (onani Supplementary Table 4 kuti mupeze mndandanda wathunthu kuphatikizapo ma amino acid sequences) adapangidwa kukhala plasmid pT7 ndikusinthidwa kukhala E. coli BL21 (DE3).E. coli yokhala ndi ma plasmids opangidwa ndi injiniya adayikidwa mu msuzi wa Luria wowonjezeredwa ndi kanamycin (70 mg/l) ndipo amakula usiku wonse pa 30 ° C ndi 250 rpm.Chikhalidwecho chinalowetsedwa 1/100 mu LB sing'anga yokhala ndi kanamycin ndikukula pa 30 ° C ndi 110 rpm mpaka OD600 itafika 0.8.Pa maphunziro a NMR, mabakiteriya amakula mu M9 yaying'ono sing'anga yokhala ndi 2 g ya D-glucose 13C (Aldrich) ndi 1 g ya ammonium chloride 15N (Cambridge Isotope Laboratories, Inc.) polemba mapuloteni okhala ndi isotopu.Chepetsani kutentha kufika madigiri 20 Celsius ndipo yambitsani puloteni ndi 0.15 mM isopropylthiogalactopyranoside (final concentration).Pambuyo pa kufotokoza kwa mapuloteni usiku wonse, maselo amakololedwa pa 7278 × g, 4 ° C kwa 20 min.Maselo a cell adayimitsidwanso mu 20 mM Tris-HCl, pH 8, ndi kuzizira mpaka kugwiritsidwa ntchito kwina.Maselo a Thawed adapangidwa pogwiritsa ntchito chosokoneza ma cell (TS series machines, Constant Systems Limited, England) pa 30 kPa.Kenako ma lysates anali centrifuged pa 25,000 g kwa mphindi 30 pa 4 ° C.Pakuti NTMiSp, ndi pellet ndiye resuspended mu 2 M urea, 20 mm Tris-HCl, pH 8, ndi sonicated 2 min (2 s pa/off, 65%), ndiye centrifuged kachiwiri pa 25,000 xg, 4° C. mkati 30 min.Wopambana kwambiri adakwezedwa pagawo la Ni-NTA, otsukidwa ndi 20 mM Tris-HCl, 2 mM imidazole, pH 8, ndipo pamapeto pake mapuloteni adachotsedwa ndi 20 mM Tris-HCl, 200 mM imidazole, pH 8. Kupanga NT2RepCT ndi NTCT, thrombin digestion imayambitsa malo (ThrCleav) pakati pa Ake ndi NT.Malo a Thrombin cleavage amapezekanso mu His-NT-ThrCleav-2Rep (amapanga 2Rep), His-thioredoxin-ThrCleav-NT (amapanga NT), His-thioredoxin-ThrCleav-CT (amapanga CT), His-Thioredoxin-ThrCleav-NT .* (amapanga NT*), His-Thioredoxin-ThrCleav-NTA72R (amapanga NTA72R), His-Thioredoxin-ThrCleav-NTFlSp (amapanga NTF1Sp), ndi His-Sulphur Redoxin-ThrCleav-NTMiSp (amapanga NTMiSp).Zomangamangazo zinaphwanyidwa ndi thrombin (1: 1000) ndi dialyzed usiku wonse ku 4 ° C. ndi 20 mM Tris-HCl, pH 8, pogwiritsa ntchito Spectra / Por dialysis membrane yokhala ndi molekyulu yolemetsa ya 6-8 kDa.Pambuyo pa dialysis, yankho limakwezedwa pagawo la Ni-NTA ndipo zotayira zomwe zili ndi mapuloteni achidwi amatengedwa.Kuchulukira kwa mapuloteni kunatsimikiziridwa poyesa kuyamwa kwa UV pa 280 nm pogwiritsa ntchito kutha kwa puloteni iliyonse, kupatulapo NTF1Sp, yomwe idagwiritsa ntchito kuyesa kwa Bradford molingana ndi ndondomeko ya wopanga.Kuyera kudatsimikiziridwa ndi SDS polyacrylamide (4-20%) gel electrophoresis ndi Coomassie brilliant blue staining.Mapuloteni adayikidwa pogwiritsa ntchito zosefera za centrifuge (VivaSpin 20, GE Healthcare) pa 4000 xg ndi 10 kDa molecular weight cutoff mumizere ya mphindi 20.
Thirani madzi a puloteni ndipo mosamala pipet 150 µl mu 1 ml yomveka bwino septum vial (8 x 40 mm Thermo Scientific).Machubuwo anatsekeredwa ndi kusindikizidwa ndi parafilm kuti asafufutike.Zitsanzo (n = 3) zidakulungidwa pa 37 ° C kapena 60 ° C ndipo nthawi zina zimatembenuzidwa kuti ziwone gelation.Zitsanzo zomwe zinalibe gel osakaniza zidakulungidwa kwa sabata imodzi.Chepetsani zomangira za NTMiSp disulfide ndi 10 mM DTT pa 10 µM mapuloteni.Kusanthula ma gelation a zokutira za silika za kangaude, kangaude wa Sweden adadulidwa, ma glands akulu akulu akulu akulu awiri adayikidwa mu 200 μl ya 20 mM Tris-HCl buffer pH 8 ndikudulidwa kuti ❖ kuyanika kupatukana ndi tiziwalo timene timatulutsa..Zomwe zili mu glands zimasungunuka mu buffer, 50 µl pofuna kudziwa kulemera kowuma (polowetsa mbale zotseguka pa 60 ° C mpaka kulemera kosalekeza) ndi 150 μl kwa gelation pa 37 ° C.
Kuyeza kwa geometry/chida kumapangidwa ndi chitsulo chosapanga dzimbiri pogwiritsa ntchito mbale yofananira yokhala ndi mainchesi 20 mm ndi kusiyana kwa 0.5 mm.Tenthetsani chitsanzo kuchokera pa 25 °C kufika pa 45 °C ndi kubwerera ku 25 °C pamlingo wa 1 °C pamphindi pogwiritsa ntchito chitsulo chosapanga dzimbiri pansi pa mbale ya Peltier.Miyezo ya vibrational inkachitika pafupipafupi 0.1 Hz ndi mzere wa viscoelastic wa zinthuzo pazovuta za 5% ndi 0.5% pazitsanzo za 100 mg/mL ndi 300-500 mg/mL, motero.Gwiritsani ntchito chipinda chosungiramo chinyezi kuti musamawuke nthunzi.Zambiri zidawunikidwa pogwiritsa ntchito Prism 9.
Kusonkhanitsa infuraredi (IR) sipekitiramu kutentha firiji kuchokera 800 mpaka 3900 cm-1.Chipangizo cha ATR, komanso njira yowunikira kudzera mu spectrometer, imatsukidwa ndi mpweya wowuma wowuma musanayambe komanso panthawi yoyesera.Mayankho (500 mg/mL kuti achepetse kuchuluka kwa mayamwidwe amadzi) adaponyedwa pamakristali, ndipo ma gels (500 mg/mL) adapangidwa asanayezedwe ndikusamutsidwa ku makhiristo (n = 3).Zithunzi za 1000 zinalembedwa ndi chisankho cha 2 cm-1 ndi zero duty cycle ya 2. Chotsatira chachiwiri chinawerengedwa pogwiritsa ntchito OPUS (Bruker) pogwiritsa ntchito njira yosalala ya mfundo zisanu ndi zinayi.Mawonekedwewa adasinthidwa kukhala gawo limodzi lophatikizana pakati pa 1720 ndi 1580 cm-1 pogwiritsa ntchito F. Menges "Spectragryph - Optical Spectroscopy Software".Mu spectroscopy ya ATR-IR, kuzama kwa mtengo wa infrared mu zitsanzo kumadalira nambala ya mafunde, zomwe zimapangitsa kuyamwa mwamphamvu pamanambala otsika kuposa manambala apamwamba.Zotsatirazi sizinakonzedwenso pazithunzi zomwe zikuwonetsedwa mu Mkuyu.3 chifukwa ndi ochepa kwambiri (Supplementary Fig. 4).Mawonedwe okonzedwa a chiwerengerochi adawerengedwa pogwiritsa ntchito pulogalamu ya Bruker OPUS.
M'malo mwake, kuwerengetsa kokwanira kwa mapuloteni opangidwa ndi mapuloteni kumatheka pambuyo pa kusinthika kodalirika kwa zigawo zomwe zili mkati mwa nsonga ya amide I.Komabe, zopinga zina zimabuka pochita.Phokoso mu sipekitiramu limatha kuwoneka ngati nsonga (zabodza) panthawi ya deconvolution.Kuphatikiza apo, pachimake chifukwa cha kupindika kwamadzi kumagwirizana ndi malo a amide I nsonga ndipo ikhoza kukhala ndi kukula kofanana kwa zitsanzo zomwe zili ndi madzi ochulukirapo, monga gel wamadzi omwe amaphunziridwa pano.Choncho, sitinayesere kuwononga kwathunthu nsonga ya amide I, ndipo zomwe tikuwona ziyenera kuganiziridwa pothandizira njira zina monga NMR spectroscopy.
Mayankho a 50 mg/ml NT ndi His-NT2RepCT adapangidwa usiku wonse pa 37°C.Kenako hydrogel idasungunuka ndi 20 mM Tris-HCl (pH 8) mpaka 12.5 mg/ml, kugwedezeka bwino ndikumapopedwa kuti aswe gel.Kenaka, hydrogel inachepetsedwa ka 10 ndi 20 mM Tris-HCl (pH 8), 5 μl ya chitsanzo inagwiritsidwa ntchito pa gridi yamkuwa yokutidwa ndi formvar, ndipo chitsanzo chowonjezera chinachotsedwa ndi pepala lopukuta.Zitsanzo zidatsukidwa kawiri ndi 5 µl ya madzi a MilliQ ndikudetsedwa ndi 1% uranyl formate kwa mphindi 5.Chotsani madontho ochulukirapo ndi pepala loyamwa, kenaka muwumitse mauna.Kujambula kunkachitika pamaguluwa pogwiritsa ntchito FEI Tecnai 12 Spirit BioTWIN yogwira ntchito pa 100 kV.Zithunzizo zinajambulidwa pa x 26,500 ndi x 43,000 magnifications pogwiritsa ntchito Veleta 2k × 2k CCD kamera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).Pachitsanzo chilichonse (n = 1), zithunzi za 10-15 zinalembedwa.ImageJ (https://imagej.nih.gov/) idagwiritsidwa ntchito posanthula zithunzi ndi kuyeza ma diameter a ulusi (n = 100, ulusi wosiyanasiyana).Prism 9 idagwiritsidwa ntchito poyesa mayeso a t-awiri (amichira iwiri).Ma fiber ake a His-NT2RepCT ndi NT anali 11.43 (SD 2.035) ndi 7.67 (SD 1.389) nm, motsatana.Nthawi yodalirika (95%) ndi -4.246 mpaka -3.275.madigiri a ufulu = 198, p <0.0001.
80 µl ya zitsanzo zamadzimadzi zomwe zili ndi 10 µM thioflavin T (ThT) zinayesedwa katatu (n = 3) pansi pazikhalidwe zosasunthika pogwiritsa ntchito mbale za Corning 96-zakuda pansi zomveka bwino (Corning Glass 3881, USA).Kusiyana kwa Fluorescence kunajambulidwa pogwiritsa ntchito fyuluta yosangalatsa ya 440 nm ndi fyuluta ya 480 nm emission (FLUOStar Galaxy yochokera ku BMG Labtech, Offenburg, Germany).Chizindikiro cha ThT sichinakhutitsidwe kapena kuzimitsidwa, monga kuyesa kosiyanasiyana kwa ThT kunachitika popanda kusintha mphamvu ya siginecha.Jambulani kuyamwa pa 360 nm poyezera utsi.Poyesera kubzala, ma gels 100 mg/mL adapangidwa pa 37 ° C., adayimitsidwanso, ndipo amagwiritsidwa ntchito kubzala pamlingo wa 5%, 10%, ndi 20%.Zambiri zidawunikidwa pogwiritsa ntchito Prism 9.
Thaw katundu wa His-NT2RepCT ndi NT>100 mg/mL pa ayezi ndi kusefa kudzera mu fyuluta ya 0.22 µm.Kuyikirako kunawerengedwa poyesa kuyamwa kwa 280 nm pogwiritsa ntchito Nanodrop.M'zitsime za mbale 96 zakuda zosamangirira (Corning) zokhala ndi pansi momveka bwino, zitsanzo zinachepetsedwa kukhala 20 mg/ml mu 20 mM Tris-HCl pH 8 ndikusakaniza ndi 5 μM ThT (concentration yomaliza), chiwerengero chonse cha zitsanzo. 50 μl mphamvu.Zitsanzo zinkajambulidwa mphindi 10 zilizonse pa 37 ° C pa microscope ya CellObserver (Zeiss) yokhala ndi njira yowunikira yowunikira komanso chisangalalo cha FITC ndi seti zosefera zowonera ThT.Lens ya 20x/0.4 imagwiritsidwa ntchito pojambula.Zen Blue (Zeiss) ndi ImageJ (https://imagej.nih.gov/) adagwiritsidwa ntchito posanthula zithunzi.Ma gels adakonzedwanso kuchokera ku NT ndi His-NT2RepCT solutions pamlingo wa 50 mg/mL wokhala ndi 20 mM Tris pH 8 ndi 5 µM ThT ndikuyikidwa pa 37 ° C kwa 90 min.Zidutswa za gel osakaniza zidasamutsidwira ku chitsime chatsopano chokhala ndi 20 mM Tris, pH 8, ndi 5 μM ThT mu mbale yosamangiriza yakuda 96 yomveka bwino pansi.Pezani fluorescence wobiriwira ndi zithunzi zowala zakumunda pa 20x/0.4 magnification.ImageJ idagwiritsidwa ntchito posanthula zithunzi.
Solution NMR spectra inapezedwa pa 310 K pa 600 MHz Bruker Avance Neo spectrometer yokhala ndi QCI Quadrupole Resonance Pulsed Gradient Field Cryoprobe (HFCN).Zitsanzo za NMR zomwe zili ndi 10 mg/mL mapuloteni osakanikirana olembedwa ndi 13C, 15N, osungunuka mu 20 mM Tris-HCl (pH 8), 0.02% (w/v) NaN3, 5% DO (v/v), (n = 1) .Kusintha kwa Chemical kwa NT2RepCT pa pH 6.7 kunagwiritsidwa ntchito popereka nsonga 23 mu 2D sipekitiramu ya 15N-HSQC.
Magic angle spinning solid NMR (MAS) mawonekedwe a 13C, 15N-olembedwa ma hydrogels adajambulidwa pa Bruker Avance III HD spectrometer pa 800 MHz yokhala ndi 3.2 mm 13C/15N{1H} chofufuzira chopanda ma elekitironi.Kutentha kwachitsanzo kunayendetsedwa pogwiritsa ntchito mpweya wosinthasintha wa kutentha kwa 277 K. Awiri-dimensional dipole rotational resonance (DARR) 76 ndi radio frequency reconnection (RFDR) 77 spectra anapezedwa pa MAS mafupipafupi a 12.5 kHz ndi 20 kHz, motero.Cross polarization (CP) kuchokera ku 1H kupita ku 13C inkagwiritsidwa ntchito pogwiritsa ntchito mzere wochokera ku 60.0 mpaka 48.0 kHz pa 1H, 61.3 / 71.6 kHz pa 13C (pa 12.5 / 20 kHz MAS) ndi nthawi yolumikizana ndi 0.5-1 ms.Spinal6478 decoupling pa 73.5 kHz idagwiritsidwa ntchito posonkhanitsa deta.Nthawi yogula inali 10 milliseconds ndipo kuchedwa kwa kuzungulira kunali masekondi 2.5.Malumikizidwe amodzi a Cα / Cβ omwe amawonedwa mu mawonekedwe a RFDR adaperekedwa motengera mawonekedwe amtundu wotsalira wamankhwala osinthika komanso kulumikizana kochulukirachulukira mu mawonekedwe a DARR.
Zipper79 database (https://services.mbi.ucla.edu/zipperdb/) idagwiritsidwa ntchito kuyesa mayendedwe a flutter ndi mphamvu ya Rosetta ya NT, NTFlSp, ndi NTMiSp.Dongosolo la Zipper limaphatikiza Rosetta Energy80, yomwe imaphatikiza ntchito zingapo zaulere zaulere kuti ziwonetsere ndikusanthula kapangidwe ka mapuloteni.Kuchuluka kwa mphamvu -23 kcal / mol kapena kutsika kumasonyeza chizolowezi chapamwamba cha fibrillate.Mphamvu zotsika zimatanthauza kukhazikika kwa zingwe ziwiri za β mu zipper conformation.Kuphatikiza apo, algorithm ya Waltz idagwiritsidwa ntchito kulosera zigawo za amyloidogenic mu NT, NTFlSp ndi NTMiSp Ref.81. (https://waltz.switchlab.org/).
The NT protein solution idasakanizidwa ndi 2-(N-morpholino)ethanesulfonic acid (MES) buffer pa pH 5.5 ndi 6.0 kutsitsa pH mpaka pH 6 ndi 7, motsatana.Mapuloteni omaliza anali 100 mg/ml.
Kuyeza kunachitika pa J-1500 CD spectrometer (JASCO, USA) pogwiritsa ntchito 300 μL cuvette ndi njira ya kuwala ya 0.1 cm.Mapuloteni adachepetsedwa kukhala 10 μM (n = 1) mu 20 mM phosphate buffer (pH 8).Kusanthula kukhazikika kwa mapuloteni pamaso pa mchere, mapuloteni adawunikidwa pamndandanda womwewo (n = 1) mu 20 mM phosphate buffer (pH 8) yomwe ili ndi 154 mM NaF kapena NaCl, motsatana.Kutentha kwa kutentha kunalembedwa pa 222 nm kuchokera ku 25 ° C mpaka 95 ° C ndi kutentha kwa 1 ° C / min.Gawo la mapuloteni opangidwa mwachilengedwe adawerengedwa pogwiritsa ntchito fomula (KDmeasure - KDfinal)/(KDstart - KDfinal).Kuphatikiza apo, mawonedwe asanu adajambulidwa pachitsanzo chilichonse kuchokera pa 260 nm mpaka 190 nm pa 25 ° C ndipo pambuyo pa kutentha mpaka 95 ° C.Mawonekedwe asanu adasinthidwa, kuwongoleredwa ndikusinthidwa kukhala molar ellipticity.Zambiri zidawunikidwa pogwiritsa ntchito Prism 9.
Kuchuluka kwa fluorescence kwa His-NT-GFP (300 mg/mL, 80 µL) kudayezedwa katatu (n = 3) mu mbale za Corning za 96-well pansi zowonekera zakuda (Corning Glass 3881, USA) pansi pazikhalidwe zosasunthika.Yezerani zitsanzo ndi chowerengera cha mbale cha fluorescence chokhala ndi kutalika kwa mafunde a 395 nm ndikujambulitsa mpweyawo pa 509 nm isanakwane ndi maola awiri pambuyo pake pa 37 ° C.Zambiri zidawunikidwa ndi Prism 9.
Purine nucleoside phosphorylase ntchito yoyesa zida (njira ya fluorometric, Sigma Aldrich) idagwiritsidwa ntchito molingana ndi malangizo a wopanga.Kuti muyese zochitika mu ma gels ndi mayankho omwe ali ndi His-NT-PNP, sakanizani 10 ng ya His-NT-PNP ndi 100 mg/mL NT ku voliyumu yonse ya 2 µL chifukwa gel osakaniza adapereka chizindikiro pamwamba pa nthawi yodziwikiratu.Kuwongolera kwa ma gels ndi mayankho opanda His-NT-PNP adaphatikizidwa.Miyezo idachitika kawiri (n = 2).Zochitazo zitayezedwa, zomwe zimasakanikirana zimachotsedwa ndipo gel osakaniza adajambulidwa kuti awonetsetse kuti gel osasunthika pakuyezera.Zambiri zidawunikidwa pogwiritsa ntchito Prism 9.
Kuti mumve zambiri za kapangidwe ka maphunziro, onani nkhani ya Nature yolumikizidwa ndi nkhaniyi.
Zithunzi 1 ndi 2 zimapereka deta yoyambirira.1c, 2a–c, 3a, b, e–g, 4, 5b, d, f, ndi 6, Nkhuyu Zowonjezera.3, mkuyu wowonjezera.5a, d, mkuyu wowonjezera.6 ndi mkuyu wowonjezera.8. Deta ya Deta yochokera mu kafukufukuyu ili mu nkhokwe ya Zenodo https://doi.org/10.5281/zenodo.6683653.Deta ya NMR yomwe idapezedwa mu kafukufukuyu idatumizidwa kumalo osungirako a BMRBig pansi pa ID yolowera bmrbig36.Mapangidwe a GFP ndi PNP adatengedwa kuchokera ku PDB (GFP 2B3Q, PNP 4RJ2).
Rising, A. ndi Johansson, J. Kupiza kangaude wochita kupanga.Mtengo wa National Chemical.biology.11, 309-315 (2015).
Babb, PL ndi al.Gulu la Nephila clavipes genome likuwonetsa kusiyanasiyana kwa majini a silika wa akangaude komanso mawonekedwe ake ovuta.National Genette.49, 895–903 (2017).