Adireshin da ake da shi a yanzu: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Cibiyar Na'urar Hotunan Halittu ta Lantarki.
Tsarin tattara haske na cibiyar amsawa 1 (RC-LH1) shine babban ɓangaren photosynthesis na ƙwayoyin cuta masu launin shuɗi. Mun gabatar da tsarin microscopy guda biyu na cryo-electron na hadaddun RC-LH1 daga Rhodopseudomonas palustris. Tsarin ƙuduri na 2.65-Å na hadaddun RC-LH114-W ya ƙunshi madaukai 14 na LH1 da ke kewaye da RC, wanda furotin W ke katsewa, yayin da hadaddun ba tare da furotin-W ba cikakken tsarin RC ne kewaye da RC. An rufe madaukai 16 na ƙaramin LH1. Kwatanta waɗannan tsarin yana ba da haske game da yanayin quinone a cikin hadaddun RC-LH1, gami da canje-canjen da ba a tantance ba a baya lokacin da aka ɗaure quinone a wurin RC QB, da kuma wurin wuraren ɗaure quinone na taimako, wanda ke taimakawa wajen wuce su zuwa RC. Tsarin furotin na W na musamman yana hana rufe madaukai na LH1, ta haka ne ke ƙirƙirar hanyar haɓaka musayar quinone/quinolone.
Makamashin da photosynthesis ke bayarwa zai iya ci gaba da rayuwa a duniya, kuma yana da babban damar amfani da fasahar hasken rana. Yayin da yake haɓaka photosynthesis a duniya, ƙwayoyin cuta masu launin shuɗi masu launin shuɗi suma suna nuna nau'ikan makamashi daban-daban da ƙarfin metabolism. Suna iya guje wa photosynthesis kuma suna girma yayin da ƙwayoyin cuta masu launin shuɗi a cikin duhu, suna iya daidaita nitrogen da carbon dioxide, suna samar da hydrogen, da kuma lalata mahaɗan aromatic (1-3). Domin samar da makamashi ga waɗannan hanyoyin, dole ne a canza haske cikin sauri da inganci zuwa makamashin sinadarai. Wannan tsari yana farawa lokacin da hadaddun eriya mai kama haske ya sha haske kuma ya canja wurin kuzarin da aka kama zuwa cibiyar amsawa (RC), ta haka ne ke fara rabuwar caji (4-7). Babban sashin photosynthesis a cikin ƙwayoyin cuta masu launin shuɗi ya ƙunshi nau'in RC na 2, wanda ke kewaye da hadaddun girbin haske 1 (LH1), yana samar da hadaddun RC-LH1 na tsakiya. An samar da LH1 ta hanyar jerin heterodimers masu lanƙwasa na αβ, kowannensu yana ɗaure ƙwayoyin cuta guda biyu (BChl) da carotenoids ɗaya ko biyu (8-12). Eriya mafi sauƙi ta LH1 ta ƙunshi heterodimers αβ guda 16 ko 17 waɗanda ke kewaye da RC (9-13) a cikin madauki mai rufewa, amma a cikin wasu hadaddun abubuwa na asali, peptides na transmembrane suna katse ci gaban LH1 da ke kewaye, Ta haka ne ke haɓaka yaduwar quinol/quinone tsakanin RC da cytochrome bc1 complex (11, 13-15). Shuke-shuken phototrophic mai launin shunayya Rhodopseudomonas (Rps.) wani samfuri ne wanda zai iya fahimtar kuzari da canja wurin electron wanda ke tallafawa photosynthesis. Tsarin lu'ulu'u na farko na Rps. Tsarin hadaddun palustris RC-LH1 shine RC, wanda ke kewaye da madaukai 15 na LH1 heterodimeric, waɗanda wani furotin da ba a sani ba wanda ake kira "Protein W" ya katse su (14). Daga baya an gano Protein-W a matsayin RPA4402, wanda ba shi da halayyar furotin 10.5kDa tare da helices guda uku da aka annabta na transmembrane (TMH) (16). Mun ba da shawarar sake suna furotin W na kwayar halittar rpa4402 zuwa pufW don ya yi daidai da sunaye da ake amfani da su don kwayoyin halittar da ke ƙunshe da RC-L, M (pufL, pufM) da LH1α, β (pufA, pufB). Abin sha'awa, furotin-W yana nan ne kawai a cikin kusan kashi 10% na RC-LH1, wanda ke nuna Rps. palustris yana samar da hadaddun RC-LH1 guda biyu daban-daban. A nan, muna bayar da rahoton tsarin cryo-EM (cryo-EM) mai ƙuduri mai girma na hadaddun guda biyu, ɗaya tare da furotin W da heterodimers αβ 14, ɗayan kuma ba tare da furotin W da madauri 16 na Heterodimer LH1 ba. Tsarinmu yana wakiltar canji a matakin fahimtar hadaddun RC-LH1 na Rps. palustris, saboda mun yi nazarin yawan kowane nau'in kuma muna da isasshen ƙuduri don sanya kowane peptide da pigments masu ɗaure da lipids da quinones masu alaƙa a sarari. Kwatanta waɗannan tsare-tsare ya nuna cewa sunadaran TMH guda uku-W waɗanda ba a samu a cikin wani hadaddun RC-LH1 ba zuwa yanzu suna samar da hanyar quinone don hanzarta musayar quinone/quinolone. An gano wasu wuraren ɗaure lipid da quinone da aka kiyaye, kuma mun bayyana sabon canji na tsari bayan haɗakar quinone da RC, wanda zai iya dacewa da photosystem II (PSII) RC na ƙwayoyin halitta masu ɗauke da iskar oxygen. Bincikenmu yana ba da sabbin fahimta game da yadda ake ɗaure quinone/quinolone da musayar a cikin hadaddun RC-LH1 na ƙwayoyin cuta masu launin shuɗi.
Domin sauƙaƙe yin cikakken bincike kan hadaddun abubuwa guda biyu da aka samu a cikin Rps. palustris, mun ware kowace RC-LH1 ta hanyar hanyoyin sinadarai. An tsarkake hadaddun sunadarai masu ƙarancin furotin (wanda daga baya ake kira ΔpufW) daga nau'in da ba shi da kwayar halittar pufW (16), kuma za a iya samar da hadaddun RC-LH1 guda ɗaya kawai. Ana samar da hadaddun furotin mai ɗauke da W ta hanyar nau'in. Ana gyara furotin W na wannan nau'in da alamar His 10x a ƙarshen C, don haka za a iya haɗa hadaddun furotin mai ɗauke da W yadda ya kamata tare da mafi yawan furotin W da ba shi da shi ta hanyar hana ƙarfe motsi. An raba hadaddun yadda ya kamata (16) Affinity Chromatography (IMAC).
Kamar yadda aka nuna a Hoto na 1, duka hadaddun suna ɗauke da ƙananan raka'a guda uku na RC (RC-L, RC-M da RC-H) waɗanda aka kewaye da eriya ta LH1. Tsarin 2.80-A na hadaddun da ba shi da furotin-W yana nuna heterodimers αβ 16, suna samar da madauri mai rufewa na LH1 wanda ke kewaye da RC gaba ɗaya, wanda daga baya ake kira da hadaddun RC-LH116. Tsarin 2.65Å na hadaddun da ke ɗauke da furotin-W yana da heterodimer 14-heterodimer LH1 wanda furotin-W ya katse, wanda daga nan ake kira RC-LH114-W.
(A da B) Wakiltar saman mahaɗin. (C da D) Alamun da aka haɗa a cikin sanduna. (E da F) Haɗaɗɗun da aka gani daga saman cytoplasmic suna da peptides da ƙananan LH1 da aka wakilta a cikin zane-zane, kuma an ƙidaya su a hannun agogo daga rafin furotin-W [wanda ya yi daidai da lambar Rba. hadaddun sphaeroides (13)]. Ga LH1-α, launin ƙaramin furotin rawaya ne; ga LH1-β, launin ƙaramin furotin shuɗi ne; ga furotin-W, furotin ja ne; ga RC-H, cyan ne; ga RC-L, orange ne; ga RC-M, magenta. Ana wakiltar cofactors da sanduna, kore yana wakiltar ƙwayoyin BChl da BPh a, shunayya yana wakiltar carotenoids, kuma rawaya yana wakiltar ƙwayoyin UQ10. (G da H) Ra'ayin da aka ɗaukaka na ratar furotin-W a cikin yankin daidai na hadaddun RC-LH114-W (G) da hadaddun RC-LH116 (H). Ana nuna abubuwan haɗin gwiwa a cikin siffar cike sarari, ana nuna quinone mai siffar chelated da shuɗi. Ana nuna gibin furotin-W ta hanyar layin shuɗi mai siffar (G), kuma ƙananan ramuka inda quinone/quinolol ke yaɗuwa akan zoben LH116 ana haskaka su da layin baƙi mai siffar (H).
Siffa ta 1 (A da B) ta nuna RC da ke kewaye da jerin LH1αβ heterodimers a buɗe ko a rufe, kowannensu yana ɗaure BChl biyu da carotenoid ɗaya (Siffa ta 1, C da D). Nazarin da aka yi a baya sun nuna cewa Rps shine hadaddun LH1. A cikin hanyar biosynthetic na spirulina xanthin, waɗannan nau'ikan suna ɗauke da gaurayen carotenoids (17). Duk da haka, spiropyrroxanthin shine babban carotenoid kuma yawansa yana da gamsarwa. Saboda haka, mun zaɓi yin samfurin spiroxanthin a duk wuraren ɗaure LH1. Alpha da beta polypeptides TMHs ne guda ɗaya tare da yankunan waje na membrane gajere (Siffa ta 1, A, B, E, da F). Kodayake ba a lura da yawan ragowar 17 a C-terminus ba, an raba alpha polypeptide daga Met1 zuwa Ala46 a cikin duka hadaddun. An rage β polypeptide daga Gly4 zuwa Tyr52 a cikin RC-LH116, kuma daga Ser5 zuwa Tyr52 a cikin RC-LH114-W. Ba a lura da yawan ragowar N-terminal guda 3 ko 4 ko C-terminal ba (Hoto na S1). Binciken ma'aunin spectrometry na gaurayen hadaddun RC-LH1 da aka shirya daga nau'in daji ya nuna cewa yankin da ya ɓace sakamakon raba waɗannan peptides ne daban-daban (Hoto na S1 da S2). An kuma lura da tsarin N-terminal na α-Met1 (f). Binciken ya nuna cewa α-peptide ya ƙunshi ragowar fMet1 zuwa Asp42/Ala46/Ala47/Ala50, kuma β-peptide ya ƙunshi ragowar Ser2 zuwa Ala53, wanda ya yi daidai da taswirar yawan EM mai ƙarancin zafi.
Daidaito tsakanin α-His29 da β-His36 yana sa BChls fuska da fuska; kowane αβ heterodimer yana haɗuwa da maƙwabtansa don samar da madauri buɗewa (RC-LH114-W) ko madauri rufewa (RC-LH116) a kusa da RC. Tsarin launi na exciton da aka haɗa (Hoto na 1, C da D). Idan aka kwatanta da madauri na 877 nm na RC-LH114-W, canjin ja na sha 880 nm na RC-LH116 shine 3 nm (Hoto na 2A). Duk da haka, bakan dichroism na zagaye kusan iri ɗaya ne (Hoto na 2B), yana nuna cewa kodayake akwai bambanci bayyananne tsakanin madaukai buɗewa da rufewa, yanayin gida na BChls yana da kama sosai. Canjin jan sha na iya zama sakamakon raguwar motsi na zafi da ƙaruwar kwanciyar hankali akan madauri rufewa (18, 19), canjin haɗin launi wanda madauri rufewa ya haifar (20, 21), ko haɗuwa da waɗannan tasirin guda biyu (11).
(A) Bakan shaƙar Ultraviolet/visible/kusan-infrared, waɗanda aka yiwa alama da launukan da suka dace kuma aka daidaita su zuwa kololuwar BPh a 775 nm. (B) Bakan shaƙar dichroism na zagaye an daidaita shi zuwa sharar BChl a 805 nm. (C da D) Bakan shaƙar ΔA da aka zaɓa daga bakan shaƙar RC-LH114-W complex (C) da RC-LH116 complex (D) da aka warware lokaci. Don samun mafi kyawun kwatantawa, duk bakan an daidaita su zuwa ∆A na −A a 0.2 ps. (E) Yawan iskar oxidation na cytochrome c2 bayan haske a gaban yawan UQ2 daban-daban (duba Hoto na S8 don bayanai na asali). (F) A cikin ƙwayoyin da aka girma a ƙarƙashin haske mai sauƙi, matsakaici ko babban ƙarfi (10, 30 ko 300μMm-2 s-1, bi da bi), ƙananan furotin W da RC-L a cikin hadaddun da aka tsarkake da rabon membrane da aka raba. Ƙayyade matakin furotin ta hanyar electrophoresis na gel na SDS-polyacrylamide da immunoassay (duba Hoto na S9 don bayanai na asali). Ƙayyade rabon da ya yi daidai da hadaddun RC-LH114-W da aka tsarkake. Rabon stoichiometric na RC-L zuwa furotin-W na hadaddun shine 1:1.
BChls a matsayi na 1 a cikin madauki na αβ14 mai nakasa na RC-LH114-W (Hoto na 1, A, C, da E) sun fi kusa da mai ba da gudummawa na farko na RC (P) da 6.8Å fiye da BChls masu daidai a cikin RC-LH116 (Hoto na 1, B, D, da F, da Hoto na S3); duk da haka, motsin sha na wucin gadi na hadaddun guda biyu ya nuna cewa ga RC-LH114-W da RC-LH116, madaidaitan lokacin canja wurin kuzarin motsawa daga LH1 zuwa RC sune 40 ±4 da 44±3 ps (Hoto na 2). , C da D, Hoto na S4 da Tebur S2). Hakanan babu wani babban bambanci a cikin canja wurin lantarki a cikin RC (Hoto na S5 da rubutun ƙarin da ke da alaƙa). Muna zargin cewa kusancin lokacin canja wurin makamashi tsakanin LH1 da RC-P ya faru ne saboda irin wannan nisa, kusurwa da kuzarin yuwuwar yawancin BChl a cikin madaukai biyu na LH1. Da alama binciken tsarin makamashin LH1 don isa ga mafi ƙarancin nisa bai fi sauri fiye da canja wurin makamashi kai tsaye daga wuraren da ba su da kyau zuwa RC ba. Madaurin LH1 mai buɗewa a cikin RC-LH114-W kuma yana iya fuskantar motsi mai zafi mara mahimmanci a ƙarƙashin yanayin zafi mai ƙarancin zafi don nazarin tsari, kuma akwai tsawon yanayin zoben αβ14 a zafin ɗaki daga nisan launi na βBChls a matsayin RC 1.
Hadakar RC-LH116 ta ƙunshi BChls 32 da carotenoids 16, kuma tsarinta gabaɗaya iri ɗaya ne da wanda aka samu daga Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 nau'in (PDB ID 7C9R) (12) da algae kore (Blc.viridis) (PDB ID 6ET5) (10). Bayan daidaitawa, an lura da ƙananan karkacewa kawai a cikin matsayin αβ heterodimers, musamman 1-5, 15, da 16 (Hoto na S6). Kasancewar furotin-W yana da tasiri mai mahimmanci akan tsarin LH1. TMHs ɗinsa guda uku an haɗa su ta hanyar gajerun madaukai, tare da N-terminal a gefen lumen na hadaddun da C-terminal a gefen cytoplasmic (Hoto na 1A da 3, A zuwa D). Protein-W galibi yana da sinadarin hydrophobic (Hoto na 3B), kuma TMH2 da TMH3 suna hulɗa da LH1αβ-14 don samar da saman membrane (Hoto na 3, B da E zuwa G). Haɗin ya ƙunshi ragowar Phe, Leu da Val a yankin membrane. Waɗannan ragowar suna da sinadarin hydrophobic amino acid da pigments αβ-14. Wasu ragowar polar kuma suna ba da gudummawa ga hulɗar, gami da haɗin hydrogen tsakanin W-Thr68 da β-Trp42 a saman ramin hadaddun (Hoto na 3, F da G). A saman cytoplasm, Gln34 yana kusa da ƙungiyar keto na carotenoids αβ-14. Bugu da ƙari, an warware kwayar n-dodecyl β-d-maltoside (β-DDM), kuma wutsiyar hydrophobic ɗinta ta faɗaɗa zuwa haɗin protein-W da αβ-14, kuma wutsiyar lipid na iya kasancewa a cikin jiki. Mun kuma lura cewa yankunan da ke warware C-terminal na furotin W da RCH suna da kusanci sosai, amma ba a cikin iyakokin ƙirƙirar takamaiman hulɗa ba (Hoto na 1, A da E). Duk da haka, akwai yiwuwar samun hulɗa a cikin amino acid na C-terminal da ba a warware su ba na waɗannan sunadarai guda biyu, wanda zai iya samar da hanyar ɗaukar furotin-W yayin haɗa hadaddun RC-LH114-W.
(A) Protein-W, wanda ke fuskantar haɗin gwiwa da LH1αβ14 a siffar zane mai ban dariya, yana da sarkar gefe mai siffar sanda (ja), wanda aka nuna a wani ɓangare na zane mai yuwuwar electrostatic (surface mai haske launin toka mai matakin siffar 0.13). (B) Protein-W wanda aka wakilta ta hanyar saman launi mai kama da hydrophobic. Ana nuna yankunan polar da caji a cyan, ana nuna yankunan hydrophobic a fari, kuma ana nuna yankunan hydrophobic a cikin orange. (C da D) Protein-W da aka wakilta a cikin zane mai ban dariya, yanayinsa iri ɗaya ne da na (A) (C), kuma ana juyawa da 180° (D). Dangane da matsayin da ke cikin jerin, ragowar da aka bambanta suna ɗaukar tsarin launin bakan gizo, inda N-terminal yake shuɗi kuma C-terminal yana ja. (E) Protein-W a cikin ra'ayi ɗaya kamar na (A), kuma ragowar da ke kan haɗin furotin-W:LH1 ana wakilta su da sanduna masu alamun da aka haɗa. (F) Ana juya Protein-W a 90° idan aka kwatanta da (E) da LH1αβ14 a cikin wakilcin zane mai zane, kuma dangane da ragowar haɗin gwiwa a cikin wakilcin sandar. An yiwa ragowar da suka rataye daga beta polypeptide lakabi. An nuna cofactor a matsayin sandar da ta dace da launin Hoto na 1, an nuna β-DDM da ya ruɓe a launin toka, kuma an nuna iskar oxygen a ja. (G) An juya ra'ayin da ke cikin (F) a 180°, tare da ragowar da aka fi sani na alpha polypeptide mai lakabi.
Protein-W yana maye gurbin αβ heterodimer (na 15 a Hoto na 1F), ta haka yana hana rufe madauki da karkatar da heterodimers guda uku na farko na αβ. An lura cewa matsakaicin kusurwar karkata na heterodimer na farko na αβ-1 idan aka kwatanta da yanayin fim ɗin shine 25° zuwa 29° (Hoto na 1, A da E), wanda aka samar ta hanyar karkata αβ-1 daga 2° zuwa 8° a cikin RC A mai kaifi-LH116 (Hoto na 1, B da F). Heterodimers na biyu da na uku suna karkata a 12° zuwa 22° da 5° zuwa 10°, bi da bi. Saboda toshewar RC, karkatawar αβ-1 ba ta haɗa da na biyu na αβ ba (wanda ya yi daidai da αβ na 16 a Hoto na 1F), don haka suna samar da rata bayyananne a cikin zoben LH1 (Hoto na 1, A da E). Saboda rashin heterodimers guda biyu na αβ, tare da asarar BChl guda huɗu da carotenoids guda biyu, babu ɗaya daga cikin carotenoids da ke ɗaure zuwa ƙaramin αβ-1 mai jujjuyawa, wanda ya haifar da zoben LH114-W wanda ke ɗauke da carotenoids 13 na masu cin ganyayyaki da kuma BChls 28. Kimanta ƙudurin gida na hadaddun abubuwa guda biyu a cikin yankuna αβ1 zuwa 7 sun yi ƙasa da na sauran madauki na LH1, wanda zai iya nuna ƙarfin da ke cikin sashin LH1 da ke kusa da wurin RC QB (Hoto na 4).
An nuna hotunan RC-LH114-W (A da B) da RC-LH116 (C da D) daga saman/gefen hoton (A da B) (A da C) da kuma saman rami na Hoto na 1. (B da D). Maɓallan masu launi suna nuna a dama.
Sauran hadaddun siffa guda ɗaya tilo da ke da rabon stoichiometric na 1:14 shine Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Duk da haka, furotin W da PufX ba su da wani tsari na musamman, kuma suna da tasiri mai mahimmanci akan tsarin LH1 nasu. PufX TMH guda ɗaya ne tare da yankin cytoplasmic na N-terminal wanda ke hulɗa da gefen cytoplasmic na sashin RC-H (13) a matsayin da ya dace da Rps. palustris LH116αβ-16. PufX yana ƙirƙirar tashar don musayar quinone/quinolone tsakanin RC-LH1 da hadaddun cytochrome bcl kuma yana nan a cikin duk hadaddun Rba. sphaeroides na tsakiya (13). Kodayake hanyar haɗin monomer-monomer tana cikin Rba. Sphaeroides RC-LH1-PufX dimer yana nan a wurin ɗaure furotin W a cikin RC-LH114-W, kuma gibin da PufX da furotin-W suka haifar yana daidai da matsayi (Hoto na S7A). Gibin da ke cikin RC-LH114-W kuma yana daidai da tashar quinone ta hasashe (8) na Pseudomonas rosea LH1, wanda peptides waɗanda ba su da alaƙa da furotin W ko PufX suka samar (Hoto na S7B). Bugu da ƙari, tashar quinone a cikin Blc. LH1 kore mai launin emerald wanda aka samar ta hanyar cire wani ƙaramin γ (7) yana cikin matsayi iri ɗaya (Hoto na S7C). Duk da cewa sunadaran daban-daban suna shiga tsakani, bayyanar waɗannan tashoshin quinone/quinolol a cikin matsayi ɗaya a cikin hadaddun RC-LH1 ya zama misali na juyin halitta mai haɗuwa, yana nuna cewa gibin da furotin W ya ƙirƙira na iya aiki azaman tashar quinone.
Gibin da ke cikin madaurin LH114-W yana ba da damar samar da yankin membrane mai ci gaba tsakanin sararin ciki na hadaddun RC-LH114-W da babban membrane (Hoto na 1G), maimakon haɗa sassan biyu ta hanyar ramin furotin kamar yadda yake a cikin furotin. Hadakar RC-LH116 tana kama da hadadden Tch. mai kama da allura (22) (Hoto na 1H). Tunda yaduwar quinone ta cikin membrane ya fi sauri fiye da yaduwar ta hanyar kunkuntar hanyar furotin, madaurin LH114-W da aka buɗe zai iya ba da damar juyawar RC cikin sauri fiye da madaurin LH116 da aka rufe, kuma yaduwar quinone zuwa cikin RC na iya zama mafi ƙuntatawa. Domin gwada ko furotin W yana shafar juyawar quinones ta hanyar RC, mun yi gwajin oxidation na cytochrome akan wani yawan ubiquinone 2 (UQ2) (kamar UQ10 na halitta tare da gajeriyar wutsiya isoprene) (Hoto na 2E). Duk da cewa kasancewar chelated quinone yana hana tantance daidaiton ma'aunin Michaelis (RC-LH114-W da RC-LH116 sun dace da 0.2±0.1μM da 0.5±0.2μM, bi da bi), matsakaicin ƙimar RC-LH114-W (4.6±0.2 e-RC-1 s-1) ya fi RC-LH116 girma da kashi 28±5% (3.6±0.1 e-RC-1 s-1).
Da farko mun kiyasta cewa furotin-W yana cikin kusan kashi 10% na hadaddun ƙwayoyin halitta (16); a nan, ƙimar zama na ƙwayoyin girma masu ƙarancin haske, matsakaici-haske, da masu yawan haske sune 15±0.6%, 11±1% da 0.9±0.5, bi da bi % (Hoto na 2F). Kwatanta adadi na mass spectrometry ya nuna cewa ƙara alamar histidine bai rage yawan furotin-W da aka kwatanta da nau'ikan nau'ikan daji ba (P = 0.59), don haka waɗannan matakan ba kayan tarihi bane na furotin-W da aka gyara (Hoto na S10). Duk da haka, wannan ƙarancin zama na furotin-W a cikin hadaddun RC-LH1 na iya ba da damar wasu RCs su juya a cikin sauri, ta haka yana rage jinkirin musayar quinone/quinolone a cikin hadaddun RC-LH116. Mun lura cewa ƙimar zama na haske mai yawa bai dace da bayanan transcriptomics na baya-bayan nan ba, wanda ke nuna cewa bayyanar kwayar halittar pufW tana ƙaruwa a ƙarƙashin haske mai ƙarfi (Hoto na S11) (23). Bambancin da ke tsakanin rubutun pufW da haɗa furotin-W cikin hadaddun RC-LH1 yana da rikitarwa kuma yana iya nuna tsarin hadaddun furotin.
A cikin RC-LH114-W, an ware cardiolipin 6 (CDL), phosphatidylcholine 7 (POPC), phosphatidylglycerol 1 (POPG) da 29 β-DDM kwayoyin halitta kuma an yi musu ƙirar CDL 6, POPC 24, POPG 2 da βDDM 12. RC-LH116 (Hoto na 5, A da B). A cikin waɗannan tsarin guda biyu, CDL kusan yana kan gefen cytoplasmic na hadaddun, yayin da POPC, POPG da β-DDM galibi suna kan gefen haske. An ware lipid da sabulun wanki guda biyu a yankin αβ-1 zuwa αβ-6 na hadaddun RC-LH114-W (Hoto na 5A), kuma an ware biyar a yankin daidai na RC-LH116 (Hoto na 5B). An sami ƙarin lipids a ɗayan gefen hadaddun, galibi CDL, waɗanda aka tara tsakanin RC da αβ-7 zuwa αβ-13 (Hoto na 5, A da B). Sauran lipids da sabulun wanke-wanke da aka warware a tsari suna nan a wajen zoben LH1, kuma sarƙoƙin acyl da aka warware sosai suna faɗaɗa tsakanin ƙananan sassan LH1, waɗanda aka ƙaddara a matsayin β-DDM a cikin RC-LH114-W, kuma an ayyana su a matsayin β-DDM a cikin cakuda RC A na β-DDM da POPC-LH116. Matsayi iri ɗaya na chelating lipids da sabulun wanke-wanke a cikin tsarinmu yana nuna cewa su wurare ne masu alaƙa da ilimin halittar jiki (Hoto na S12A). Matsayin ƙwayoyin da suka yi daidai a cikin Tch suma suna da kyakkyawan daidaito. Mai laushi da Trv. Nau'in 970 RC-LH1s (Hoto na S12, B zuwa E) (9, 12) da ragowar haɗin hydrogen na rukunin kan lipid sun nuna kyakkyawan kiyayewa a cikin jeri (Hoto na S13), yana nuna cewa CDL da aka kiyaye wanda ke ɗaure zuwa RC (24), waɗannan wuraren na iya kasancewa a cikin hadaddun RC-LH1.
(A da B) An wakilta peptides na RC-LH114-W (A) da RC-LH116 (B) ta hanyar zane-zane, kuma ana wakiltar launukan da sanduna, ta amfani da tsarin launi a Hoto na 1. An nuna lipids a ja, kuma an nuna sabulun wanki a launin toka. UQ da aka ɗaure zuwa wuraren RC QA da QB rawaya ne, yayin da UQ da aka ware yake shuɗi. (C da D) Ra'ayoyi iri ɗaya kamar (A) da (B), tare da cire lipids. (E zuwa G) Babban ra'ayi na Q1(E), Q2(F) da Q3(G) daga RC-LH116, tare da sarƙoƙi na gefe waɗanda ke tasiri ga juna. An nuna haɗin hydrogen a matsayin layukan baƙi masu lanƙwasa.
A cikin RC-LH116, duka RC QA da QB UQ, waɗanda ke shiga cikin canja wurin lantarki a cikin tsarin rabuwar caji, an wargaza su a wuraren da suka ɗaure. Duk da haka, a cikin RC-LH114-W, quinone na QB ba a warware shi ba kuma za a tattauna dalla-dalla a ƙasa. Baya ga quinones na QA da QB, an ware ƙwayoyin UQ guda biyu masu lanƙwasa (wanda ke tsakanin zoben RC da LH1) a cikin tsarin RC-LH114-W bisa ga ƙungiyoyin kai da suka warware sosai (wanda ke cikin Q1 da Q2, bi da bi). Sarari na 5C). An sanya raka'a biyu na isoprene zuwa Q1, kuma taswirar yawa tana warware cikakkun wutsiyoyin isoprene 10 na Q2. A cikin tsarin RC-LH116, an warware ƙwayoyin UQ10 guda uku masu lanƙwasa (Q1 zuwa Q3, Figure 5D), kuma duk ƙwayoyin suna da yawan haske a cikin wutsiya (Hoto na 5, D zuwa G). A cikin tsarin guda biyu, matsayin ƙungiyoyin kan quinone na Q1 da Q2 suna da kyakkyawan daidaito (Hoto na S12F), kuma suna hulɗa ne kawai da RC. Q1 yana wurin ƙofar rata ta W na RC-LH114-W (Hoto na 1G da 5, C, D da E), kuma Q2 yana kusa da wurin ɗaure QB (Hoto na 5, C, D) da F). Rassan L-Trp143 da L-Trp269 da aka kiyaye suna kusa da Q1 da Q2 kuma suna ba da damar hulɗar π mai yuwuwa (Hoto na 5, E da F, da Hoto na S12). L-Gln88, 3.0 Å daga iskar oxygen ta nesa na Q1, yana ba da haɗin hydrogen mai ƙarfi (Hoto na 5E); wannan ragowar yana cikin duk RCs banda dangantakar da ta fi nisa (Hoto na S13). An maye gurbin L-Ser91 da Thr a mafi yawan sauran RCs (Hoto na S13), shine 3.8 Angstroms daga methyl oxygen na Q1, kuma yana iya samar da raunin haɗin hydrogen (Hoto na 5E). Q3 ba ya da alaƙa ta musamman, amma yana cikin yankin hydrophobic tsakanin sashin RC-M da sashin LH1-α na 5 zuwa 6 (Hoto na 5, D da G). Q1, Q2 da Q3 ko quinones na kusa an kuma warware su a cikin Tch. Gentle, Trv. Strain 970 da Blc. Tsarin iris (9, 10, 12) yana nuna wurin ɗaure quinone na taimako da aka kiyaye a cikin hadaddun RC-LH1 (Hoto na S12G). UQ guda biyar da aka rusa a cikin RC-LH116 sun yi daidai da 5.8±0.7 na kowace hadadden da aka ƙayyade ta hanyar babban aikin ruwa chromatography (HPLC), yayin da UQ guda uku da aka rusa a cikin RC-LH114-W sun yi ƙasa da ƙimar da aka auna ta 6.2±0.3 (Hoto na S14) yana nuna cewa akwai ƙwayoyin UQ da ba a warware su ba a cikin tsarin.
Polypeptides na L da M na pseudo-symmetric kowannensu yana ɗauke da TMH guda biyar kuma suna samar da heterodimer wanda ya haɗu da dimer BChl guda ɗaya, monomers na BChl guda biyu, monomers na bacteriophage guda biyu (BPh), da kuma ƙarfe ɗaya wanda ba Heme ba da ƙwayoyin UQ10 ɗaya ko biyu. Ta hanyar kasancewar haɗin hydrogen akan ƙungiyar ketone mai ƙarewa da kuma tarinsa da aka sani a cikin Rps, an haɗa carotenoids a cikin M-subunit, wanda aka sanya wa suna cis-3,4-dehydroorhodopin. Nau'in (25). Yankin membrane na waje na RC-H an haɗa shi da membrane ta hanyar TMH guda ɗaya. Tsarin RC gabaɗaya yayi kama da RC na ƙananan nau'i uku na nau'ikan da ke da alaƙa (kamar Rba). sphaeroides (PDB ID: 3I4D). Macrocycles na BChl da BPh, kashin baya na carotenoid da baƙin ƙarfe mara heme suna haɗuwa a cikin kewayon ƙuduri na waɗannan tsarin, kamar yadda ƙungiyar shugaban UQ10 a wurin QA da quinone na QB a RC-LH116 (Hoto na S15).
Samuwar tsarin RC guda biyu tare da adadin zama a wurin QB daban-daban yana ba da sabuwar dama don bincika canje-canje masu daidaituwa tare da ɗaure quinone na QB. A cikin hadaddun RC-LH116, quinone na QB yana cikin matsayi na "kusa" (26), amma rabuwar RC-LH114-W ba shi da quinone na QB. Babu quinone na QB a cikin RC-LH114-W, wanda abin mamaki ne saboda hadaddun yana aiki, fiye da hadaddun RC-LH116 tare da quinone na QB da aka warware a cikin tsari. Kodayake zoben LH1 guda biyu suna yin kusan quinones shida, biyar an warware su a cikin tsarin zoben RC-LH116 da aka rufe, yayin da uku kawai ke da iyaka a cikin tsarin zoben RC-LH114-W da aka buɗe. Wannan karuwar rashin lafiyar tsarin na iya nuna saurin maye gurbin wuraren RC-LH114-W QB, saurin kinetics na quinone a cikin hadaddun, da kuma ƙaruwar yuwuwar ketare madauki na LH1. Muna ba da shawarar cewa rashin UQ a ​​wurin RC QB na RC-LH114-W na iya zama sakamakon wani hadadden tsari mai rikitarwa da aiki, kuma wurin QB na RC-LH114-W an daskare shi nan da nan a cikin juyawar UQ. Mataki na musamman (an rufe ƙofar shiga wurin QB) yana nuna yanayin wannan aikin.
Ba tare da QB ba, juyawar L-Phe217 mai zuwa zuwa wani matsayi wanda bai dace da ɗaurewar UQ10 ba, domin zai haifar da karo na sarari da naúrar isoprene ta farko ta wutsiya (Hoto na 6A). Bugu da ƙari, manyan canje-canje a bayyane suke a bayyane, musamman helix de (gajeren helix a cikin madauki tsakanin TMH D da E) inda aka canza L-Phe217 zuwa aljihun ɗaurewar QB da juyawar L-Tyr223 (Hoto na 6A) Don karya haɗin hydrogen tare da tsarin M-Asp45 da rufe ƙofar wurin ɗaurewar QB (Hoto na 6B). Helix de yana juyawa a tushensa, Cα na L-Ser209 an canza shi da 0.33Å, yayin da L-Val221Cα an canza shi da 3.52Å. Babu canje-canje da za a iya gani a cikin TMH D da E, waɗanda ba za a iya jurewa ba a cikin duka tsarin (Hoto na 6A). Kamar yadda muka sani, wannan shine tsari na farko a cikin RC na halitta wanda ke rufe wurin QB. Kwatantawa da cikakken tsarin (wanda aka ɗaure da QB) ya nuna cewa kafin a rage quinone, ana buƙatar canji mai kama da juna don sanya shi shiga cikin quinone. L-Phe217 yana juyawa don samar da hulɗar π-stacking tare da rukunin kan quinone, kuma helix yana juyawa waje, yana ba da damar kwarangwal na L-Gly222 da sarkar gefe na L-Tyr223 su samar da hanyar sadarwa ta haɗin hydrogen tare da tsarin haɗin hydrogen mai karko (Hoto na 6, A da C).
(A) Zane mai kama da hologram (sarkar L, sarkar orange/M, magenta) da kuma tsarin apo (launin toka), inda aka nuna maɓallan ragowar a cikin siffar wakilcin sanda. An wakilta UQ10 da sandar rawaya. Layin mai dige-dige yana nuna haɗin hydrogen da aka samar a cikin dukkan tsarin. (B da C) Wakiltar saman apolipoprotein da tsarin zobe gaba ɗaya, yana nuna iskar oxygen na sarkar gefe na L-Phe217 a cikin shuɗi da L-Tyr223 a cikin ja, bi da bi. Rabon L shine lemu; rabon M da H ba a launi su ba. (D da E) Apolipoprotein (D) da kuma rabon (E) RC QB [launi ta (A) bi da bi] da Thermophilus thermophilus PSII (kore, shuɗi tare da quinone na filastik; PDB ID: 3WU2) Daidaita (58).
Ba zato ba tsammani, kodayake akwai wasu gine-gine na RCs marasa QB ba tare da LH1 ba, ba a bayar da rahoton canje-canjen siffar da aka lura a cikin wannan binciken a baya ba. Waɗannan sun haɗa da tsarin rage girman QB daga Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) da Rba. sphaeroides (PDB ID: 1OGV) (29), waɗanda duk kusan iri ɗaya ne da tsarin QB ɗinsu gaba ɗaya. Dubawa kusa da 3PRC ya nuna cewa ƙwayoyin sabulun LDAO (Lauryl Dimethyl Amine Oxide) suna ɗaure a ƙofar wurin QB, wanda zai iya hana sake tsarawa zuwa yanayin rufewa. Kodayake LDAO ba ya ruɓewa a wuri ɗaya a cikin 1EYS ko 1OGV, waɗannan RCs an shirya su ta amfani da sabulu iri ɗaya don haka suna iya haifar da irin wannan tasirin. Tsarin lu'ulu'u na Rba. Sphaeroides RC wanda aka haɗa shi da cytochrome c2 (PDB ID: 1L9B) shi ma da alama yana da wurin QB da aka rufe. Duk da haka, a wannan yanayin, yankin N-terminal na polypeptide na RC-M (yana hulɗa da wurin ɗaure QB ta hanyar haɗin H na ragowar Tyr akan helix na Q) yana ɗaukar yanayin da ba na halitta ba, kuma ba a ƙara bincika canjin yanayin QB ba (30). Abin da ke kwantar da hankali shi ne cewa ba mu ga irin wannan nakasar polypeptide na M a cikin tsarin RC-LH114-W ba, wanda kusan iri ɗaya ne da yankin N-terminal na RC-LH116 RC. Ya kamata kuma a lura cewa bayan kawar da eriya ta LH1 mai tushen sabulu, an warware RCs na apolipoprotein a cikin PDB, wanda ya kawar da wuraren quinone na ciki da lipids a cikin rata tsakanin RC da saman ciki na zoben LH1 da ke kewaye (31, 32). RC ya ci gaba da aiki saboda yana riƙe da dukkan abubuwan haɗin gwiwa, sai dai quinone na QB mai narkewa, wanda ba shi da ƙarfi kuma sau da yawa ana ɓacewa yayin tsarin shiri (33). Bugu da ƙari, an san cewa cire LH1 da lipids na halitta daga RC na iya yin tasiri ga ayyuka, kamar gajeriyar rayuwar yanayin P+QB da aka raba da caji (31, 34, 35). Saboda haka, muna hasashen cewa wanzuwar zoben LH1 na gida da ke kewaye da RC na iya kiyaye wurin QB "rufe", ta haka ne ke kiyaye yanayin gida kusa da QB.
Duk da cewa apolipoprotein (ba tare da QB quinone ba) da cikakken tsarin suna wakiltar hotuna biyu ne kawai na juyawar wurin QB, maimakon jerin abubuwan da suka faru, akwai alamun cewa ana iya ɗaurewa don hana sake ɗaurewa ta hanyar hydroquinone. Don hana hana substrate. Hulɗar quinolol da quinone kusa da wurin QB na apolipoprotein na iya bambanta, wanda ke haifar da RC ta ƙi shi. An daɗe ana ba da shawarar cewa canje-canje na tsari suna taka rawa a cikin ɗaurewa da rage quinones. Ikon RCs masu daskarewa don rage quinones bayan daidaitawar duhu ya lalace (36); X-ray crystallography ya nuna cewa wannan lalacewa ta faru ne saboda quinones na QB da aka kama a cikin yanayin "distal" kimanin 4.5 Å daga matsayin kusanci mai aiki (26), 37). Muna ba da shawarar cewa wannan yanayin ɗaurewa na nesa hoto ne na yanayin matsakaici tsakanin apolipoprotein da cikakken tsarin zobe, wanda ke biyo bayan hulɗar farko da quinone da buɗe wurin QB.
Nau'in RC na II da aka samu a cikin hadaddun PSII na wasu ƙwayoyin cuta masu kama da juna da kuma cyanobacteria, algae da tsire-tsire yana da kiyaye tsari da aiki (38). Daidaiton tsarin da aka nuna a Hoto na 6 (D da E) yana jaddada kamanceceniya tsakanin PSII RCs da wurin QB na hadaddun RC na ƙwayoyin cuta. Wannan kwatancen ya daɗe yana zama misali don nazarin tsarin da ke da alaƙa da ɗaure quinone da ragewa. Littattafan da suka gabata sun nuna cewa canje-canjen tsari suna tare da rage PSII na quinones (39, 40). Saboda haka, idan aka yi la'akari da kiyaye juyin halitta na RC, wannan hanyar ɗaurewa da ba a lura da ita ba a baya na iya dacewa da wurin QB na PSII RC a cikin tsire-tsire masu ɗauke da iskar oxygen.
Rps ΔpufW (sharewar pufW mara lakabi) da PufW-His (C-terminal 10x His-tagged protein-W da aka bayyana daga nau'ikan pufW locus na halitta). An bayyana palustris CGA009 a cikin aikinmu na baya (16). An samo waɗannan nau'ikan da mahaɗan nau'in isogenic na daji daga injin daskarewa ta hanyar zana ƙananan ƙwayoyin halitta akan PYE (kowane lita 5 g -1) (an adana a cikin LB a -80 °C, yana ɗauke da furotin 50% (w/v) glycerol, yeast extract da succinate) agar [1.5% (w/v)]. An sanya farantin a cikin duhu a zafin ɗaki a ƙarƙashin yanayin rashin lafiyar jiki, sannan aka haskaka shi da haske fari (~50 μmolm-2 s-1) wanda OSRAM 116-W halogen bulbs (RS Components, UK) ya samar na tsawon kwanaki 3 zuwa 5 har sai da wani yanki guda ɗaya ya bayyana. An yi amfani da wani yanki guda ɗaya don yin allurar M22+ matsakaici (41) 10 ml (41) tare da ƙarin 0.1% (w/v) casamino acid (wanda daga baya ake kira M22). An noma al'adar a ƙarƙashin ƙarancin iskar oxygen a cikin duhu a 34°C tare da girgiza a 180 rpm na tsawon awanni 48, sannan aka yi allurar 70 ml na al'adar a ƙarƙashin irin wannan yanayi na tsawon awanni 24. Ana amfani da al'adar semi-aerobic tare da girman 1 ml don yin allurar 30 ml na matsakaiciyar M22 a cikin kwalbar gilashi mai haske na duniya 30 ml kuma an yi masa allurar tare da tashin hankali (~50μmolm-2 s-1) na tsawon awanni 48 ta hanyar ƙarfin maganadisu mai hana ruwa. Sannan an yi allurar 30 ml na al'ada tare da kimanin lita 1 na al'ada a ƙarƙashin irin wannan yanayi, wanda aka yi amfani da shi don yin allurar kimanin lita 9 na al'ada da aka haskaka a ~200 μmolm-2 s-1 na tsawon awanni 72. An tattara ƙwayoyin halittar ta hanyar amfani da centrifugation a 7132 RCF na tsawon mintuna 30, sannan aka sake dasa su a cikin ~10 ml na 20 mM tris-HCl (pH 8.0), sannan aka adana su a -20°C har sai an buƙata.
Bayan narkewar ruwa, ƙara wasu lu'ulu'u na deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) da allunan Roche holoenzyme protease inhibitor guda biyu (Merck, UK) zuwa ƙwayoyin da aka sake dasawa. A cikin ƙwayar matsin lamba ta Faransa mai nauyin psi 20,000 (Aminco, Amurka), an katse ƙwayoyin sau 8 zuwa 12. Bayan cire ƙwayoyin da ba su karye ba da tarkace marasa narkewa ta hanyar centrifugation a 18,500 RCF na tsawon mintuna 15 a 4°C, an fitar da membrane daga lysate mai launin ta hanyar centrifugation a 113,000 RCF na tsawon awanni 2 a 43,000°C. A jefar da rabon da ke narkewa sannan a sake dasa membrane mai launi a cikin 100 zuwa 200 ml na 20 mM tris-HCl (pH 8.0) kuma a daidaita shi har sai babu tarin abubuwa da aka gani. An saka membrane da aka dakatar a cikin 20 mM tris-HCl (pH 8.0) (Anatrace, Amurka) wanda ke ɗauke da 2% (w/v) β-DDM na tsawon awa 1 a cikin duhu a 4°C tare da juyawa a hankali. Sai a saka centrifuge a 70°C don narke 150,000 RCF a 4°C na tsawon awa 1 don cire ragowar da ba ya narkewa.
An shafa membrane mai narkewa daga nau'in ΔpufW a kan ginshiƙin musayar ion na DEAE Sepharose 50 ml tare da juzu'i uku na ma'aunin ... Tattara bakan sha tsakanin 250 da 1000 nm, kiyaye kashi tare da rabon sha (A880/A280) fiye da 1 a 880 zuwa 280 nm, a narkar da shi sau biyu a cikin ma'ajiyar ɗaurewa, sannan a sake amfani da wannan hanyar a kan ginshiƙin tsarkakewa na DEAE. A narkar da sassan tare da rabon A880/A280 sama da 1.7 da A880/A805 sama da 3.0, a yi zagaye na uku na musayar ion, kuma a riƙe sassan tare da rabon A880/A280 sama da rabon 2.2 da A880/A805 sama da 5.0. An tattara sinadarin da aka tsarkake wanda aka rage girmansa zuwa ~2 ml a cikin matatar centrifugal ta Amicon 100,000 (MWCO), sannan aka ɗora shi a kan ginshiƙin cire girman Superdex 200 16/600 (GE Healthcare, US) wanda ke ɗauke da ma'aunin NaCl 200 mM, sannan aka cire shi a cikin ma'aunin ɗaya a 1.5 CV. Tattara ma'aunin sha na ɓangaren cire girman, sannan a tattara ma'aunin sha tare da rabon A880/A280 sama da rabon 2.4 da A880/A805 sama da rabon 5.8 zuwa 100 A880, kuma nan da nan a yi amfani da su don shirya grid ko ajiya na cryo-TEM. Ajiye a -80°C har sai an buƙata.
An shafa membrane mai narkewa daga nau'in PufW-His a kan ginshiƙin HisPrep FF Ni-NTA Sepharose mai 20 ml (20 mM tris-HCl (pH 8.0) wanda ke ɗauke da 200 mM NaCl da 0.03% (w/w)) a cikin IMAC buffer (GE Healthcare). v) β-DDM). An wanke ginshiƙin da CVs biyar na IMAC buffer, sannan da CVs biyar na IMAC buffer wanda ke ɗauke da 10 mM histidine. An cire babban haɗin daga ginshiƙin da buffers biyar na IMAC waɗanda ke ɗauke da histidine 100 mM. An tattara ɓangaren da ke ɗauke da hadaddun RC-LH114-W zuwa ~10 ml a cikin tanki mai juyawa wanda aka sanya masa matatar Amicon 100,000 MWCO (Merck, UK), an narkar da shi sau 20 da buffer mai ɗaurewa, sannan aka ƙara shi zuwa 25 ml. A ginshiƙin DEAE Sepharose, ana amfani da CVs huɗu da aka ɗaure da buffer a gaba. A wanke ginshiƙin da ma'ajiyar haɗin CV guda huɗu, sannan a cire mahaɗin a kan CV guda takwas a kan layi mai juzu'i na 0 zuwa 100 mM NaCl (a cikin ma'ajiyar haɗin), da kuma sauran CV guda huɗu da ke ɗauke da ma'ajiyar haɗin 100 mM. Sauran mahaɗan da suka fito daga sodium chloride tare da rabon A880/A280 sama da 2.4 kuma rabon A880/A805 sama da ƙashi 4.6 an tattara su zuwa ~2 ml a cikin matatar centrifugal Amicon 100,000 MWCO, kuma an cika su da IMAC 1.5 CV a gaba. Buffer ya daidaita Superdex 200 16/600 girman Buffer, sannan a cire su a cikin ma'ajiyar guda ɗaya akan CV 1.5. Tattara siginar sha na sassan da ba a iya cire girman su ba sannan a tattara siginar sha tare da rabon A880/A280 sama da rabon 2.1 da A880/A805 sama da rabon A4.6 zuwa 100 A880, waɗanda ake amfani da su nan take don shirya grid ɗin TEM daskararre ko adana su a -80°C har sai an buƙata.
An yi amfani da injin daskarewa na Leica EM GP don shirya grids na TEM masu ƙarancin zafi. An narkar da hadaddun a cikin IMAC buffer zuwa A880 na 50, sannan aka ɗora 5μl a kan sabon raga na jan ƙarfe mai rufi da carbon QUANTIFOIL 1.2/1.3 (Agar Scientific, UK). A saka grid ɗin a 20°C da kuma ɗanɗanon da ya dace da kashi 60% na daƙiƙa 30, sannan a goge shi ya bushe na daƙiƙa 3, sannan a kashe shi a cikin ruwa mai ethane a -176°C.
An yi rikodin bayanan hadaddun RC-LH114-W akan eBIC (Cibiyar Binciken Halittu ta Lantarki) (Tushewar Hasken Diamond na Burtaniya) tare da na'urar hangen nesa ta Titan Krios, wacce ke aiki a ƙarfin lantarki mai sauri na 300kV, tare da girman girma na 130,000 × da kuzari na - Zaɓi tazara ta 20 eV. An yi amfani da Gatan 968 GIF Quantum tare da na'urar gano kololuwar K2 don yin rikodin hotuna a yanayin ƙidaya don tattara bayanai. Girman pixel ɗin da aka daidaita shine 1.048Å, kuma ƙimar allurar shine 3.83 e-Å-2s-1. Ya tattara fim ɗin a cikin daƙiƙa 11 kuma ya raba shi zuwa sassa 40. Yi amfani da yankin da aka rufe da carbon don sake mayar da hankali kan na'urar hangen nesa, sannan ya tattara fina-finai uku a kowane rami. Jimilla, an tattara fina-finai 3130, tare da ƙimar defocus tsakanin -1 da -3μm.
An tattara bayanan hadaddun RC-LH116 ta amfani da wannan na'urar hangen nesa a dakin gwaje-gwaje na Asterbury Biostructure (Jami'ar Leeds, Burtaniya). An tattara bayanan a yanayin ƙidaya tare da ƙara girman 130 k, kuma an daidaita girman pixel zuwa 1.065 Å tare da allurar 4.6 e-Å-2s-1. An yi rikodin fim ɗin cikin daƙiƙa 12 kuma an raba shi zuwa sassa 48. Jimilla, an tattara fina-finai 3359, tare da ƙimar defocus tsakanin -1 da -3μm.
Ana gudanar da dukkan ayyukan bayanai a cikin bututun Relion 3.0 (42). Yi amfani da Motioncorr 2 (43) don gyara motsin haske ta hanyar auna yawan allurai, sannan yi amfani da CTFIND 4.1 (44) don tantance sigar CTF (bambancin aikin canja wurin bambanci). An nuna alamun hoto na yau da kullun bayan waɗannan matakan farko na aiki a cikin Hoto na 2. S16. Ana samar da samfurin zaɓi na atomatik ta hanyar zaɓar kusan pixels 250 na barbashi 1000 da hannu a cikin firam ɗin pixel 250 kuma babu rarrabuwa mai girma biyu (2D), don haka ana ƙin waɗannan rarrabuwa waɗanda suka dace da gurɓataccen samfurin ko kuma ba su da halaye da za a iya gani. Sannan, an yi zaɓin atomatik akan duk ƙananan hotuna, kuma RC-LH114-W ya kasance barbashi 849,359, kuma hadaddun RC-LH116 ya kasance barbashi 476,547. Duk ƙwayoyin da aka zaɓa sun yi zagaye biyu na rarrabuwar 2D mara ma'ana, kuma bayan kowace gudu, ana ƙin ƙwayoyin da suka dace da yankin carbon, gurɓatar samfurin, babu wasu siffofi masu bayyana ko ƙwayoyin da suka yi karo da juna, wanda ke haifar da 772,033 (90.9%) da 359,678 (75.5%) Ana amfani da ƙwayoyin cuta don rarraba RC-LH114-W da RC-LH116 a cikin 3D bi da bi. An samar da samfurin ma'ana na farko na 3D ta amfani da hanyar saukowa mai sauƙi. Ta amfani da samfurin farko a matsayin ma'ana, an rarraba ƙwayoyin da aka zaɓa zuwa rukuni huɗu a cikin 3D. Ta amfani da samfurin a cikin wannan rukuni a matsayin ma'ana, yi gyaran 3D akan ƙwayoyin da ke cikin babban rukuni, sannan yi amfani da matattarar low-pass ta farko ta 15Å don rufe yankin narkewa, ƙara pixels 6 na gefuna masu laushi, kuma bayan aiwatar da pixels ɗin don gyara aikin canja wurin Modulation na kololuwar Gatan K2 na na'urar gano saman. Ga bayanan RC-LH114-W, an gyara wannan samfurin farko ta hanyar cire ƙarfin yawa a gefunan abin rufe fuska (wanda aka cire daga babban yawan hadaddun a cikin UCSF Chimera). Ana amfani da samfuran da suka haifar (ƙudurin RC-LH114-W da RC-LH116 sune 3.91 da 4.16 Å, bi da bi) azaman nuni ga zagaye na biyu na rarrabuwar 3D. An haɗa ƙwayoyin da aka yi amfani da su cikin ajin farko na 3D kuma ba su ƙunshi alaƙa mai ƙarfi da unguwa ba. Haɗuwa ko rashin fasalulluka na tsari bayyanannu. Bayan zagaye na biyu na rarrabuwar 3D, an zaɓi nau'in da ke da mafi girman ƙuduri [Ga RC-LH114-W, rukuni ɗaya shine ƙwayoyin 377,703 (44.5%), ga RC-LH116, akwai nau'i biyu, jimillar ƙwayoyin 260,752 (54.7%) , Inda suke iri ɗaya ne kawai lokacin da aka daidaita bayan juyawar farko tare da ƙaramin bambanci]. An sake fitar da ƙwayoyin da aka zaɓa a cikin akwati mai girman pixel 400 kuma an gyara su ta hanyar gyaran 3D. Ana samar da abin rufe fuska na solvent ta amfani da matattarar farko ta 15Å mai ƙarancin wucewa, faɗaɗa taswirar pixel 3 da abin rufe fuska mai laushi na pixel 3. Ta amfani da gyaran CTF na kowace barbashi, gyaran motsi na kowace barbashi da zagaye na biyu na gyaran CTF na kowace barbashi, gyaran 3D, rufewar solvent da kuma bayan sarrafawa ana yin su bayan kowane mataki don ƙara inganta yanayin da ya haifar. Ta amfani da ƙimar yankewa ta FSC (Fourier Shell Correlation Coefficient) na 0.143, ƙudurin samfuran ƙarshe na RC-LH114-W da RC-LH116 sune 2.65 da 2.80Å, bi da bi. An nuna lanƙwasa FSC na samfurin ƙarshe a Hoto na 2. S17.
An sauke dukkan jerin sunadaran daga UniProtKB: LH1-β (PufB; UniProt ID: Q6N9L5); LH1-α (PufA; UniProtID: Q6N9L4); RC-L (PufL; UniProt ID: O83005); RC-M (PufM; UniProt ID: A0A4Z7); RC-H (PuhA; UniProt ID: A0A4Z9); Protein-W (PufW; UniProt ID: Q6N1K3). An yi amfani da SWISS-MODEL (45) don gina samfurin RC, wanda ya ƙunshi jerin sunadaran RC-L, RC-M da RC-H kuma an yi amfani da tsarin lu'ulu'u na Rba. sphaeroides RC azaman samfuri (PDB ID: 5LSE) (46). Yi amfani da kayan aikin "tsarin daidaitawa" a cikin UCSF Chimera don daidaita samfurin da aka samar zuwa taswirar (47), inganta tsarin furotin, da cofactor [4×BChl a (sunan residue name na monomer = BCL), 2×BPh a (BPH), nau'i ɗaya ko biyu na UQ10 (U10), ƙarfe ɗaya mara heme (Fe) da ɗaya 3,4-dihydrohexacarbonylcholine (QAK)] yi amfani da Coot (48) don ƙarawa. Tunda QAK ba ya samuwa a cikin ɗakin karatu na monomer, an daidaita shi ta amfani da kayan aikin eLBOW a cikin PHENIX (49).
Na gaba, an gina ƙaramin rukunin LH1. Da farko, an yi amfani da kayan aikin gini na atomatik a cikin PHENIX (49) don gina wani ɓangare na jerin LH1 ta atomatik ta amfani da taswirar da jerin furotin na LH1-α da LH1-β a matsayin shigarwa. Zaɓi ƙaramin rukunin LH1 mafi cikakken, cire shi kuma ɗora shi cikin Coot, ƙara jerin da ya ɓace da hannu, sannan a gyara dukkan tsarin da hannu kafin a ƙara BCls a (BCL) guda biyu da spirilloxanthin (CRT) [bisa ga yawan Rps na hadaddun LH1 da abubuwan da aka sani na carotenoid. Nau'in (17)]. Kwafi cikakken sashin LH1, kuma yi amfani da "Kayan Taswirar Docking" na UCSF Chimera don sanyawa a yankin da ba na samfurin ba na yawan LH1, sannan a tace shi a cikin Coot; maimaita tsarin har sai an yi wa dukkan ƙananan sassan LH1 ƙira. Don tsarin RC-LH114-W, ta hanyar cire yawan da ba a raba ba a cikin Coot, ana raba furotin daga sauran abubuwan da ba su da furotin a cikin taswirar USCF Chimera kuma ana amfani da kayan aikin Autobuild don kafa samfurin farko, da sauran ƙananan sassa (protein-W). A cikin PHENIX (49). Ƙara duk wani jerin da ya ɓace zuwa samfurin da aka samo a cikin Coot (48), sannan a gyara dukkan ƙananan sassa da hannu. Sauran yawan da ba a raba ba ya dace da haɗin lipids (ID na ɗakin karatu na PDB monomer na CDL = CDL, POPC = 6PL da POPG = PGT), sabulun β-DDM (LMT) da ƙwayoyin UQ10 (U10). Yi amfani da inganta PHENIX (49) da ingantawa da hannu a cikin Coot (48) don kammala cikakken samfurin farko har sai ba za a iya ƙara inganta ƙididdigar samfurin da ingancin gani na dacewa ba. A ƙarshe, yi amfani da LocScale (50) don kaifafa taswirar gida, sannan a yi wasu zagayowar yin kwaikwayon yawan da ba a raba ba da ingantawa ta atomatik da hannu.
An nuna nau'ikan peptides, cofactors da sauran lipids da quinones da aka haɗa a cikin yawansu a cikin Hoto na 1 da 2. S18 zuwa S23. Bayanan kididdiga na samfurin ƙarshe an nuna su a cikin Tebur S1.
Sai dai idan an ƙayyade wani abu daban, an tattara spectra na sha UV/Vis/NIR akan na'urar auna haske ta Cary60 (Agilent, Amurka) a tazara ta 1 nm daga 250 nm zuwa 1000 nm da lokacin haɗakarwa na 0.1s.
A narkar da samfurin a cikin wani yanki mai siffar quartz tare da hanyar 2 mm zuwa A880 na 1, sannan a tattara siginar sha tsakanin 400 da 1000 nm. An tattara siginar dichroic mai zagaye akan na'urar spectropolarimeter ta Jasco 810 (Jasco, Japan) a tazara tsakanin 400 nm da 950 nm a ƙimar scan na 20 nm min-1.
Ana tantance ma'aunin ƙarewar molar ta hanyar rage hadaddun tsakiya zuwa A880 na kimanin 50. A narkar da ƙarar 10μl a cikin ma'aunin ɗaure 990μl ko methanol, sannan a tattara ma'aunin sha nan da nan don rage lalacewar BChl. An ƙididdige abun ciki na BChl na kowane samfurin methanol ta hanyar ma'aunin ɓacewa a 771 nm na 54.8 mM-1 cm-1, kuma an ƙayyade ma'aunin ɓacewa (51). Raba yawan BChl da aka auna da 32 (RC-LH114-W) ko 36 (RC-LH116) don tantance yawan haɗuwar tsakiya, wanda daga nan ake amfani da shi don tantance bakan sha na samfurin iri ɗaya da aka tattara a cikin ma'aunin ƙarewa na buffer. a layi ɗaya. An ɗauki ma'auni guda uku da aka maimaita ga kowane samfurin, kuma an yi amfani da matsakaicin sha na matsakaicin BChl Qy don lissafi. Ma'aunin ƙarewar RC-LH114-W da aka auna a 878 nm shine 3280±140 mM-1 cm-1, yayin da ma'aunin ƙarewar RC-LH116 da aka auna a 880 nm shine 3800±30 mM-1 cm-1.
An auna UQ10 bisa ga hanyar da ke cikin (52). A takaice, an yi amfani da tsarin Agilent 1200 HPLC na juyawa (RP-HPLC). A narke kimanin nmol na RC-LH116 ko RC-LH114-W a cikin 50μl na 50:50 methanol:chloroform wanda ke ɗauke da 0.02% (w/v) ferric chloride, sannan a yi allurar Beckman Coulter Ultrasphere ODS 4.6 mm. A narke a cikin 1 ml-1 min-1 a 40°C a cikin HPLC solvent (80:20 methanol:2-propanol) a kan ginshiƙi ×25 cm. A yi isocratic exlution a cikin HPLC solvent don sa ido kan sha a 275 nm (UQ10), 450 nm (carotenoids) da 780 nm (BChl) na tsawon awa 1. An haɗa kololuwar da ke cikin chromatogram na 275 nm a minti 25.5, wanda bai ƙunshi wasu mahaɗan da za a iya ganowa ba. Ana amfani da yankin da aka haɗa don ƙididdige adadin molar na UQ10 da aka cire tare da lanƙwasa daidaitawa da aka ƙididdige daga allurar tsarkakkun ma'auni daga 0 zuwa 5.8 nmol (Hoto na S14). An yi nazarin kowane samfurin a cikin kwafi uku, kuma kuskuren da aka ruwaito ya yi daidai da SD na matsakaicin.
An shirya wani maganin da ke ɗauke da hadaddun RC-LH1 tare da matsakaicin sha na Qy na 0.1 tare da rage yawan μM na zuciya na doki c2 (Merck, UK) da 0 zuwa 50 μMUQ2 (Merck, UK). An shirya samfuran 1-ml guda uku a kowane taro na UQ2 kuma an saka su cikin duhu cikin dare a 4°C don tabbatar da cikakken daidaitawa da duhu kafin a auna su. An ɗora maganin a cikin na'urar spectrophotometer mai motsi ta OLIS RSM1000 wacce aka sanye da layin wuta/layi na 300 nm, mashigar 1.24 mm, 0.12 mm na tsakiya da 0.6 mm. An sanya matatar wucewa mai tsawon nm 600 a ƙofar bututun samfurin da bututun mai amfani da hoto don cire hasken motsawa. An sa ido kan sha a 550 nm tare da lokacin haɗa 0.15 s. Ana fitar da hasken motsawa daga LED mai ƙarfin 880 nm M880F2 (Diode Mai Fitar da Haske) (Thorlabs Ltd., UK) ta hanyar kebul na fiber optic a ƙarfin 90% ta hanyar mai sarrafa DC2200 (Thorlabs Ltd., UK) kuma ana fitar da shi zuwa tushen haske a kusurwar 90° na. Hasken aunawa yana adawa da madubi don dawo da duk wani haske da samfurin bai sha ba da farko. Kula da shan iska na daƙiƙa 10 kafin hasken daƙiƙa 50. Sannan an ƙara sa ido kan shan iska na daƙiƙa 60 a cikin duhu don tantance girman yadda quinolol ke rage cytochrome c23 + ba tare da ɓata lokaci ba (duba Hoto na S8 don bayanai na asali).
An sarrafa bayanan ta hanyar daidaita ƙimar farko mai layi tsakanin daƙiƙa 0.5 zuwa 10 (ya danganta da yawan UQ2) da kuma matsakaicin ƙimar dukkan samfuran uku a kowane yawan UQ2. An yi amfani da yawan RC-LH1 da aka ƙididdige ta hanyar ma'aunin ƙarewa don canza ƙimar zuwa ingancin catalytic, wanda aka zana a cikin Origin Pro 2019 (OriginLab, Amurka), kuma an daidaita shi da samfurin Michaelis-Menten don tantance ƙimar Km da Kcat da ke bayyane.
Don auna sha na ɗan lokaci, an narkar da samfurin RC-LH1 zuwa ~2μM a cikin ma'aunin IMAC wanda ke ɗauke da sodium ascorbate 50 mM (Merck, Amurka) da Terbutin 0.4 mM (Merck, Amurka). Ana amfani da Ascorbic acid a matsayin mai ba da kyautar lantarki ta hadaya, kuma ana amfani da tert-butaclofen a matsayin mai hana QB don tabbatar da cewa babban mai ba da gudummawar RC ya rage (wato, ba a yi amfani da shi ba) a duk tsawon aikin aunawa. Ana ƙara kimanin 3 ml na samfurin a cikin ƙwayar juyawa ta musamman (kimanin 0.1 m a diamita, 350 RPM) tare da tsawon hanyar gani na 2 mm don tabbatar da cewa samfurin da ke cikin hanyar laser yana da isasshen lokaci don daidaitawa da duhu tsakanin bugun motsawa. Yi amfani da bugun laser ~100-fs don faɗaɗa tsarin laser Ti: Sapphire (Spectra Physics, Amurka) don tayar da samfurin a 880 nm a ƙimar maimaitawa na 1 kHz (20 nJ don NIR ko 100 nJ don Vis). Kafin tattara bayanai, a fallasa samfurin a hasken motsawa na kimanin mintuna 30. Fuskar zata haifar da rashin kunna QA (mai yiwuwa rage QA sau ɗaya ko biyu). Amma a lura cewa wannan tsari zai iya canzawa saboda bayan dogon lokaci na daidaitawar duhu, RC zai koma aikin QA a hankali. An yi amfani da na'urar auna zafin jiki ta Helios (Ultrafast Systems, Amurka) don auna zafin zafin jiki na ɗan lokaci tare da jinkirin lokaci na -10 zuwa 7000 ps. Yi amfani da software na Surface Xplorer (Ultrafast Systems, Amurka) don raba saitin bayanai, sannan a haɗa su da daidaita su. Yi amfani da fakitin software na CarpetView (Light Conversion Ltd., Lithuania) don amfani da saitin bayanai da aka haɗa don samun bambancin yanayin zafi da suka shafi lalacewa, ko amfani da aikin da ya ƙunshi ƙarin haske da yawa tare da martanin kayan aiki don dacewa da juyin halittar yanayin zafi na tsawon zango ɗaya a Origin (OriginLab, Amurka).
Kamar yadda aka ambata a sama (53), an shirya wani fim mai ɗauke da sinadarin LH1 wanda ba shi da eriya ta RC da ta gefe ta LH2. An narkar da membrane ɗin a cikin tris 20 mM (pH 8.0) sannan aka ɗora shi a cikin wani yanki mai siffar quartz tare da hanyar gani ta 2 mm. An yi amfani da bugun laser na 30nJ don motsa samfurin a 540 nm tare da lokacin jinkiri na -10 zuwa 7000 ps. A sarrafa saitin bayanai kamar yadda aka bayyana don samfurin Rps. pal.
An yi wa membrane ɗin pellet ta hanyar centrifugation a 150,000 RCF na tsawon awanni 2 a 4°C, sannan aka sake haɗa shi da 880 nm a cikin 20 mM tris-HCl (pH 8.0) da 200 mM NaCl. A narke membrane ɗin ta hanyar juyawa a hankali a cikin 2% (w/v) β-DDM na tsawon awa 1 a cikin duhu a 4°C. An narkar da samfurin a cikin 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) zuwa yawan furotin na 2.5 mg ml-1 (Bio-Rad analysis). An ci gaba da sarrafa shi daga hanyar da aka buga a baya (54), inda aka fara da narkar da furotin 50 μg zuwa jimlar 50 μl TEAB wanda ke ɗauke da sodium laurate 1% (w/v) (Merck, UK). Bayan an yi amfani da sonication na tsawon daƙiƙa 60, an rage shi da 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) a zafin jiki na 37°C na tsawon mintuna 30. Don S-alkylation, a saka samfurin da 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) sannan a ƙara shi daga maganin isopropanol stock na 200 mM na tsawon mintuna 10 a zafin ɗaki. An gudanar da narkewar proteolytic ta hanyar ƙara 2 μg trypsin/endoproteinase Lys-C cakuda (Promega UK) sannan a saka a zafin jiki na 37°C na tsawon awanni 3. An cire laurate surfactant ta hanyar ƙara 50 μl ethyl acetate da 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) da kuma yin vortexing na tsawon daƙiƙa 60. An haɓaka rabuwar matakin ta hanyar amfani da centrifugation a 15,700 RCF na tsawon mintuna 5. A cewar ka'idar masana'anta, an yi amfani da wani ginshiƙi mai juyawa na C18 (Thermo Fisher Scientific, UK) don yin numfashi da kuma cire gishiri a cikin ƙananan matakin da ke ɗauke da peptide. Bayan bushewa ta hanyar ventifugation na vacuum, an narkar da samfurin a cikin 0.5% TFA da 3% acetonitrile, kuma an yi nazarin 500 ng ta hanyar nanoflow RP chromatography tare da mass spectrometry ta amfani da sigogin tsarin da aka bayyana a baya.
Yi amfani da MaxQuant v.1.5.3.30 (56) don gano furotin da ƙididdigewa don bincika bayanan Rps. palustris proteome (www.uniprot.org/proteomes/UP000001426). An ajiye bayanan mass spectrometry proteomics a cikin ProteomeXchange Alliance ta hanyar ma'ajiyar abokin hulɗa na PRIDE (http://proteomecentral.proteomexchange.org) a ƙarƙashin mai gano bayanai na PXD020402.
Don yin bincike ta hanyar RPLC tare da electrospray ionization mass spectrometry, an shirya hadaddun RC-LH1 daga nau'in Rps na daji. Ta amfani da hanyar da aka buga a baya (16), yawan furotin da aka samar a cikin ƙwayoyin palustris shine 2 mg ml-1 a ​​cikin 20 mM Hepes (pH 7.8), 100 mM NaCl da 0.03% (w/v) β- (Bio-Rad analysis)) DDM. Dangane da ka'idar masana'anta, yi amfani da kayan tsarkakewa na 2D (GE Healthcare, Amurka) don cire furotin 10 μg ta hanyar hazo, kuma a narkar da ruwan da ya faɗi a cikin 20 μl 60% (v/v) formic acid (FA), 20% (v/v) Acetonitrile da 20% (v/v) ruwa. An yi nazarin microliters biyar ta hanyar RPLC (Dionex RSLC) tare da mass spectrometry (Maxis UHR-TOF, Bruker). Yi amfani da ginshiƙin MabPac 1.2×100 mm (Thermo Fisher Scientific, UK) don rabuwa a 60°C da 100μlmin -1, tare da gradient na 85% (v / v) mai narkewa A [0.1% (v / v) FA da 0.02% (V/v) maganin ruwa TFA] zuwa 85% (v/v) mai narkewa B [0.1% (v/v) FA da 0.02% (v/v) a cikin 90% (v/v) acetonitrile TFA] Ta amfani da tushen ionization na lantarki da sigogi na tsoho na fiye da mintuna 60, mai auna taro yana samun 100 zuwa 2750 m/z (rabo-da-caji). Tare da taimakon tashar albarkatun ExPASy bioinformatics kayan aikin FindPept (https://web.expasy.org/findpept/), zana taswirar taro zuwa ƙananan sassan hadaddun.
An girma ƙwayoyin halittar na tsawon awanni 72 a ƙasa da 100 ml na NF-low (10μMm-2 s-1), matsakaici (30μMm-2 s-1) ko babba (300μMm-2 s-1). Matsakaicin M22 (M22 matsakaici inda aka cire ammonium sulfate kuma aka maye gurbin sodium succinate da sodium acetate) a cikin kwalbar sukurori mai girman milimita 100 (23). A cikin zagaye biyar na 30-s, an lulluɓe beads na gilashin micron 0.1 a rabon girma na 1:1 don lalata ƙwayoyin kuma a sanyaya su a kan kankara na tsawon mintuna 5. An cire abubuwan da ba sa narkewa, ƙwayoyin da ba su karye ba da beads na gilashi ta hanyar centrifugation a 16,000 RCF na tsawon mintuna 10 a cikin benchtop microcentrifuge. An raba membrane ɗin a cikin rotor na Ti 70.1 tare da RCF 100,000 a cikin 20 mM tris-HCl (pH 8.0) tare da gradient na sucrose 40/15% (w/w) na tsawon awanni 10.
Kamar yadda aka bayyana a cikin aikinmu na baya, gano cutar ta His tag akan PufW (16). A takaice, hadaddun tsakiya mai tsabta (11.8 nM) ko membrane wanda ke ɗauke da irin wannan yawan RC (wanda aka ƙaddara ta hanyar oxidation cire raguwar bambancin bakan da daidaita nauyin akan gel mai launi) a cikin ma'aunin loading na SDS 2x (Merck, UK) an narkar da shi sau biyu. An raba sunadaran akan gel na bis-tris NuPage 12% (Thermo Fisher Scientific, UK). An yi wa gel fenti da Coomassie Brilliant Blue (Bio-Rad, UK) don lodawa da ganin ƙaramin RC-L. An canza furotin akan gel na biyu zuwa membrane na polyvinylidene fluoride (PVDF) na methanol (Thermo Fisher Scientific, UK) don gwajin immunoassay. An toshe membrane na PVDF a cikin foda madara mai nauyin 50 mM tris-HCl (pH 7.6), NaCl 150 mM, 0.2% (v / v) Tween-20 da 5% (w / v), sannan aka haɗa shi da babban antibody na anti-His (a cikin Rage sinadarin antibody buffer [50 mM tris-HCl (pH 7.6), NaCl 150 mM da 0.05% (v/v) Tween-20] a cikin 1:1000 A190-114A, Bethel Laboratories, Amurka) na tsawon awanni 4. Bayan an wanke sau 3 na mintuna 5 a cikin maganin hana ƙwayoyin cuta, an haɗa membrane ɗin da maganin hana ƙwayoyin cuta na horseradish peroxidase (Sigma-Aldrich, UK) (wanda aka narkar da shi 1:10,000 a cikin maganin hana ƙwayoyin cuta). A saka shi a cikin injin don ba da damar ganowa (minti 5 bayan an wanke shi 3 a cikin maganin hana ƙwayoyin cuta) ta amfani da sinadarin chemiluminescence na WESTAR ETA C 2.0 (Cyanagen, Italiya) da Amersham Imager 600 (GE Healthcare, UK).
Ta hanyar zana rarrabawar ƙarfin kowane gel mai launi ko layin immunoassay, haɗa yankin da ke ƙarƙashin kololuwar da kuma ƙididdige rabon ƙarfin RC-L (gel mai launi) da Protein-W (immunoassay), a cikin ImageJ (57) Shirya hoton. An canza waɗannan rabo zuwa rabon molar ta hanyar ɗauka cewa rabon RC-L zuwa furotin-W a cikin samfurin RC-LH114-W tsarkakakke shine 1:1 kuma yana daidaita dukkan bayanan da aka saita daidai.
Don ƙarin kayan aiki don wannan labarin, da fatan za a duba http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
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David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe, Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Tsarin da ke da ƙuduri mai girma na hadaddun tarkon haske 1 a cibiyar amsawa yana ba da sabbin fahimta game da yanayin quinone.
David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe, Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Tsarin da ke da ƙuduri mai girma na hadaddun tarkon haske 1 a cibiyar amsawa yana ba da sabbin fahimta game da yanayin quinone.
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