Obwire obu†Endagiiriro eriho hati: OX11 0DE, Bungyereza, ekyombeko kya Diamond, Paaka y’eby’obuhangwa n’eby’obuhangwa eya Harwell, Dietcote, Oxfordshire, Bungyereza, Co.
Ekicweka ky’okugarukamu eky’omushana-okugyesha 1 (RC-LH1) nikyo kicweka kikuru ky’okukora eby’okurya by’omushana gw’obukooko oburikureeta eby’okurya by’omushana. Tukataho ebishushani bibiri by’okukyebera ebiri omu nkora ya RC-LH1 kuruga omu Rhodopseudomonas palustris. Omuringo gw‘okushoboorora 2.65-Å ogw‘ekicweka kya RC-LH114-W gurimu ebicweka 14 eby‘ekicweka kya LH1 ebirikwehinguririza RC, ebirikuteganisibwa puroteyini W, obwo ekicweka ekitaine puroteyini-W kiri RC yoona eyehinguririize RC. Okukinga 16 ekicweka kya LH1 orukoba. Okugyeragyeranisa eby‘obuhangwa ebi nikuha okumanya aha nkora ya quinone omu RC-LH1 complex, obariiremu empindahinduka z‘omubiri ezitakamanyirwe enyima baaba nibakwatanisa quinone aha mwanya gwa RC QB, hamwe n‘omwanya gw‘emyanya y‘okukwatiraho quinone ey‘okuhwera, ebirikuhwera Kuzihitsya ahari RC. Omuringo gw’omutaano ogwa puroteyini ya W niguzibira okukinga orukoba rwa LH1, obwo nigureetaho omuhanda gw’okutunguura okuhindura kwa quinone/quinolone.
Amashanyarazi agarikuheebwa omushana nigabaasa kugumaho haihi amagara goona omunsi, kandi gaine obushoborozi bw'amaani omu bya tekinologiya y'eizooba. N’obu baraabe nibatunguura eby’okurya by’omushana omu nsi yoona, obukooko oburikureeta eby’okurya omu mushana obw’erangi ya purple nabwo nibworeka emiringo y’amaani etari emwe n’emwe n’obushoboorozi bw’okuhindura eby’okurya. Nizibaasa kwetantara okukora eby’okurya by’omushana kandi zikure nk’obukooko oburikurya eby’okurya ebitari bimwe na bimwe omu mwirima, nizibaasa kuboneza nitrogen na carbon dioxide, zireete hydrogen, kandi zisheeshe ebirungo by’omubazi (1-3). Okubaasa kuha amaani g’emiringo egi, omushana guteekwa kuhindurwa omu bwangu kandi omu muringo murungi kuba amaani g’eby’obuhangwa. Enkora egi neetandika obu ekicweka ky’antenna ekirikukwaata omushana kirikunywa omushana kandi kitwara amaani agakwatsirwe omu mwanya gw’okugarukamu (RC), obwo kitandika okwahukana kwa charge (4 – 7). Ekicweka ky’omutaano eky’okukora eby’okurya omu bacteria z’erangi ya purple phototrophic kirimu ekika kya 2 RC, kyehinguririziibwe ekicweka ky’okusharura omushana 1 (LH1), ekirikureetaho ekicweka ky’omutaano ekya RC-LH1. LH1 neekorwaho omubaro gw’ebicweka bya αβ ebitarikushushana ebikozirwe omu nkora, buri kimwe aharibyo nikikwatanisa n’ebicweka bibiri bya bacteria chlorophyll (BChl) a molecules hamwe na carotenoids emwe nari ibiri (8-12). Antenna ya LH1 eyorobi munonga erimu αβ heterodimers 16 nari 17 ezirikwehinguririza RC (9-13) omu nkora ekingire, kwonka omu bindi bizibu by‘omutaano, peptides ezirikuraba omu nkora niziteganisa okugumizamu kwa LH1 eyehinguririize , Obwo nizitunguura okujanjaara kwa quinol/quinone na cytochme1 ahagati ya bRC1. 13-15). Ekimera ky’erangi ya papo ekirikurya ebishana Rhodopseudomonas (Rps.) n’ekihangirwe ky’eky’okureeberaho ekirikubaasa kwetegyereza amaani n’okuhingisa kw’ebiriisa ebirikushagika okukora eby’okurya. Omuringo gw’okubanza ogw’ekibiriiti ogwa Rps. Omuringo gw’ekicweka kya palustris RC-LH1 complex ni RC, eyehinguririziibwe ebicweka 15 ebya LH1 ebitarikushushana, ebirikuteganisibwa puroteyini etarikumanywa erikwetwa “Protein W” (14). Protein-W bwanyima ekamanyirwa nka RPA4402, eri puroteyini etaine mitwarize ya 10.5kDa erimu ebicweka bishatu ebirikuteberezibwa (TMH) (16). Nituteekateeka kuhindura eiziina ry’obutafaari bwa rpa4402 oburikuhandiika puroteyini W kuhindurwa pufW kuhikaana n’eiziina eririkukoresibwa aha nkora y’obutafaari oburikuhandiika RC-L, M (pufL, pufM) na LH1α, β (pufA, pufB) obucweka obukye. Ekirikushemeza, puroteyini-W neeshangwa omu bicweka nka 10% byonka ebya RC-LH1, erikworeka Rps. palustris neereetaho ebicweka bibiri bya RC-LH1 ebitarikushushana. Aha, nituha ripooti y‘omutindo gw‘ahaiguru ogwa cryo-EM (cryo-EM) gw‘ebicweka bibiri by‘omutaano, kimwe kiine puroteyini W na 14 αβ heterodimers, ekindi kitaine puroteyini W n‘ekirungo kya 16 Heterodimer LH1 ekikingire. Enkora yaitu neejwekyera empindahinduka y‘eitembezo omu kwetegyereza RC-LH1 complex ya Rps. palustris, ahabwokuba tukashwijuma omubaro gw’abantu abarikushushana ba buri nkora kandi twine okushoboorora okurikumara kuha buri peptide n’erangi ezibohirwe n’ebishaju ebiine akakwate na quinones. Okugyeragyeranisa ebicweka ebi nikworeka ngu puroteyini ishatu eza TMH-W ezitakashangirwe omu RC-LH1 complex endiijo yoona kuhitsya hati nizireetaho omuhanda gwa quinone kutunguura okuhindura kwa quinone/quinolone. Omubaro gw’emyanya y’okuteerana amashaju n’ekiriisa kya quinone ebibiikire gumanyirwe, kandi twashohoza empindahinduka nsya y’omubiri bwanyima y’okuteerana kwa quinone na RC, erikubaasa kuba nungi aha photosystem II (PSII) RC y’ebihangirwe ebirikurya ebiriisa ebirimu oxygen. Ebi twashanga nibituha okumanya okusya aha nkora y‘okukwatira hamwe n‘okuhindura kwa quinone/quinolone omu RC-LH1 core complex y‘obukooko oburikureeta ebishushani by‘erangi ya papo.
Okubaasa kuhwera omu kucondooza okuhikire aha bikwatiraine n’ebizimbe bibiri ebirikushangwa omu Rps. palustris, nitutaanisa buri RC-LH1 turikukoresa emiringo y’eby’amagara. Ekirungo kya puroteyini W-deficient complex (aha muheru ekirikumanyirwa nka ΔpufW) kikashemezibwa kuruga omu kika ekitarimu pufW gene (16), kandi ekirungo kimwe kyonka ekya RC-LH1 nikibaasa kukorwa. Ekirungo ekirimu puroteyini W nikikorwa omu muringo gw’ekika. Puroteni W ey’omuringo ogu neehindurwa n’akabonero ka 10x His aha C-terminus yaayo, kugira ngu puroteyini W-erimu complex ebaasa kugambwaho gye na puroteyini W erikukirayo obutagira puroteyini W obwo erikuremesa ekyoma kutambura. Ekizibu eki nikitaanisibwa gye (16) Okukyebera okurikukwata aha nkora (IMAC).
Nkoku kirikworekwa omu kishushani 1, ebizimbe byombi birimu ebicweka bishatu bya RC (RC-L, RC-M na RC-H) ebyetooroirwe antenna ya LH1. Omuringo gwa 2.80-A ogw‘ekizibu ekitaine puroteyini-W nigworeka αβ heterodimers 16, ezirikukora orukoba rwa LH1 orukingire orurikwehinguririza RC yoona, ahaifo nirumanyirwa aha kizibu kya RC-LH116. Omuringo gwa 2.65Å ogw’ekirungo ekirimu puroteyini-W gwine 14-heterodimer LH1 erikuteganisibwa puroteyini-W, ahaifo neeyetwa RC-LH114-W.
(A na B) Okujwekyera aha mutwe gw’ekirungo. (C na D) Erangi ezikwataniise ezirikworekwa omu nkoni. (E na F) Ebizibu ebireebirwe kuruga aha mutwe gwa cytoplasmic biine peptides na LH1 subunits ebirikworekwa omu katuuni, kandi nibibarwa omu nkora y‘eshaha kuruga omu gap ya protein-W [ebirikuhikaana n‘okubara kwa Rba. ekizibu ky’ebihaha (13)]. Aha LH1-α, erangi y’ekicweka kya puroteyini neeba eri eya kyenju; aha LH1-β, erangi y’ekicweka kya puroteyini neeba eri bururu; aha puroteyini-W, puroteyini neetukura; aha RC-H, n’eky’omubiri; aha RC-L, n’omucungwa; aha RC-M, erangi erikutukura. Cofactors nizijwekyerwa emiti, ekinyaatsi nikijwekyera molekyu za BChl na BPh a, erangi ya papo neejwekyera carotenoids, kandi eya kyenju neejwekyera molekyu za UQ10. (G na H) Okureeba okuhango kw‘ekicweka kya puroteyini-W omu kicweka ekirikushushana eky‘ekicweka kya RC-LH114-W (G) n‘ekicweka kya RC-LH116 (H). Cofactors nizoorekwa omu muringo gw‘okujwisa omwanya, chelated quinone neeyorekwa omu bururu. Empaaka ya puroteyini-W neeyorekwa omurongooti gwa bururu ogurimu obubonero omu (G), kandi ebiina ebikye ahu quinone/quinolol erikujanjaara aha mpeta ya LH116 nibyorekwa omurongooti ogurimu obubonero oburikwiragura omu (H).
Ekishushani 1 (A na B) nikyoreka RC eyehinguririziibwe enkora ezikingire nari ezikingire eza LH1αβ heterodimers, buri emwe neekwatanisa BChl ibiri na carotenoid emwe (ekishushani 1, C na D). Okucondooza okukozirwe enyima kworekire ngu Rps n‘ekicweka kya LH1. Omu muhanda gw’okukora eby’okurya ebirikwetwa spirulina xanthin, ebika ebi biine ebiriisa ebitaine mugasho (17). Kwonka spiropyrroxanthin niyo erikukirayo omu kureetaho ebiriisa kandi oburemeezi bwayo nibumara. Nahabwekyo, tukasharamu kugyeragyeranisa spiroxanthin aha myanya yoona erikukwatiraho LH1. Alpha na beta polypeptides ni TMHs zimwe eziine ebicweka by‘aheeru eby‘omubiri ebigufu (ekishushani 1, A, B, E, na F). N’obu oburemeezi bw’ebisigarira 17 aha C-terminus buraabe butareebekire, alpha polypeptide ekasharaho kuruga ahari Met1 kuhika ahari Ala46 omu bizibu byombi. β polypeptide ekakyendeera kuruga ahari Gly4 kuza ahari Tyr52 omu RC-LH116, kandi kuruga ahari Ser5 kuza ahari Tyr52 omu RC-LH114-W. Tihariho oburemeezi bw‘ebisigarira bya N-terminal 3 nari 4 nari 13 ebya C-terminal ebyareebekire (ekishushani S1). Okushwijuma kwa mass spectrometry okwa RC-LH1 complex etaburiire eyatebeekanisiibwe kuruga omu nkora y’omukibira kukareebeka ngu ekicweka ekyabuzire kikaba kiri ekirugire omu kuhendagura kw’ebicweka ebi eby’omubiri ebitarikushushana (ekishushani S1 na S2). Okukora kwa N-terminal kwa α-Met1 nakwo kukareebeka (f). Okweshwijuma kukareebeka ngu α-peptide erimu ebisigarira fMet1 kuhika Asp42/Ala46/Ala47/Ala50, kandi β-peptide erimu ebisigarira Ser2 kuhika Ala53, ekirikuhikaana gye na maapu y’obuhango bwa EM ey’obwire bukye.
Okukwatanisa kwa α-His29 na β-His36 nikureetera BChls kureebana amaisho aha maisho; buri αβ heterodimer neeteerana na bataahi baayo kukora orukoba orukingire (RC-LH114-W) nari orukoba orukingire (RC-LH116) okwehinguririize RC Omubaro gw‘erangi ogurikukwatanisa n‘eby‘okureeberaho (ekishushani 1, C na D). Kugyeragyeranisa n’ekicweka kya 877 nm ekya RC-LH114-W, okuhinduka kw’okunywa okurikwiragura okwa 880 nm okwa RC-LH116 ni 3 nm (ekishushani 2A). Kwonka, circular dichroism spectrum eri haihi kushushana (ekishushani 2B), ekirikworeka ngu n‘obu haraabe hariho entaaniso erikureebwa gye ahagati y‘enzindo ezikingire n‘ezikingire, embeera y‘omu kyanga eya BChls neeshushana munonga. Okuhinduka kw‘erangi erikutukura kw‘okunywa nikubaasa kuba nikuruga omu kukyendeera omu kugyendera kw‘okwotsya n‘okwongyera aha kuhama aha rubaju orukingire (18, 19), empindahinduka omu kugaita erangi erikureetwaho orubaju orukingire (20, 21), nari okuteerana kw‘ebintu ebi bibiri (11).
(A) Eby’okureeberaho by’okunywanisa eby’omushana/ebirikureebeka/ebiri haihi n’eby’omushana, enshonga zabyo ziine akamanyiso n’erangi zaabo ezirikuhikaana kandi zihindurwa eza butoosha kuhika aha mutwe gwa BPh ahari 775 nm. (B) Eby’okureeberaho by’okwetoorora ebiri omu nkora kuhika aha kunywanisa kwa BChl ahari 805 nm. (C na D) Ebishushani bya ΔA ebitoorainwe kuruga omu bishushani by‘okunywanisa ebyashobooroirwe omu bwire eby‘ekicweka kya RC-LH114-W (C) na RC-LH116 ekigumire (D). Okugyeragyeranisa kurungi, ebishushani byona nibihindurwa bya butoosha kuhika ahari ∆A ya −A ahari 0.2 ps. (E) Omubaro gw‘okuhinduka kwa cytochrome c2 bwanyima y‘okukyeberwa omu kubaho kw‘ebicweka bitari bimwe na bimwe bya UQ2 (reeba ekishushani S8 ahabw‘ebihandiiko ebitahikire). (F) Omu butafaari obukuzire ahansi y’omushana gw’amaani makye, ahagati nari ahaiguru (10, 30 nari 300μMm-2 s-1, buri kimwe), puroteyini W na RC-L subunits omu kizibu ekishemeziibwe n’omubaro gw’ebizimba ebitaanisiibwe. Manya omutindo gwa puroteyini orikukoresa SDS-polyacrylamide gel n‘okukyebera obutafaari bw‘omubiri (reeba ekishushani S9 ahabw‘ebihandiiko ebitahikire). Manya omubaro ogurikuhikaana n‘ekicweka kya RC-LH114-W ekishemeziibwe. Omubaro gwa stoikiyometiri ogwa RC-L na puroteyini-W ey’ekizibu ni 1:1.
BChls eziri omu mwanya 1 omu nkora ya αβ14 ehindwirwemu eya RC-LH114-W (ekishushani 1, A, C, na E) nizihika haihi n‘omuhi w‘obuhwezi w‘okubanza owa RC (P) na 6.8Å kukira BChls ezirikushushana omu RC-LH116 (ekishushani. na F, B, D); kwonka, enkora y‘okunywanisa ey‘obwire bukye ey‘ebizibu bibiri neeyoreka ngu ahari RC-LH114-W na RC-LH116, obwire bw‘okuhingisa amaani g‘okusiimura obutarikuhinduka kuruga ahari LH1 kuza ahari RC ni 40 ±4 na 44±3 ps (ekishushani 2). , C na D, Ekishushani S4 n‘Ekipande S2). Kandi tihariho ntaaniso mpango omu kuhingisa eby‘amashanyarazi omu RC (ekishushani S5 n‘ebihandiiko by‘okwongyeraho ebiine akakwate). Nituteekateeka ngu okuhikaana kw‘ahaiguru kw‘obwire bw‘okuhingisa amaani ahagati ya LH1 na RC-P nikureetwaho okushushana kw‘oburaingwa, enkokora n‘amaani agarikubaasa kubaho ga BChl nyingi omu nshonda ibiri eza LH1. Nikireebeka ngu okucondooza aha nkora y‘amaani ga LH1 kuhika aha rugendo orukye tikurikurahuka kukira okuhingisa amaani butunu kuruga omu myanya etari y‘omugasho kuza omu RC. Open-loop LH1 loop omu RC-LH114-W neebaasa n’okutunga okutambura kw’okwotsya okutari kw’omugasho ahansi y’embeera z’obutagatsi bukye ahabw’okushwijuma eby’obuhangwa, kandi hariho okutebeekanisa kw’empeta αβ14 okuraingwa aha butagasi bw’omu kishengye kuruga aha rugendo rw’okujwara βBChls omu mwanya gwa RC 1.
Ekicweka kya RC-LH116 kirimu BChls 32 na carotenoids 16, kandi entebeekanisa yaakyo yoona neeshushana n’eyatungirwe kuruga omu bihingwa ebirikwetwa Thermochromatium (Tch.) pidpidum. (12) n’ebishaka ebirikwetwa Blc.viridis (PDB ID 6ET5) (10). Bwanyima y‘okuteerana, okutaana okukye omu myanya ya αβ heterodimers kukareebeka, namunonga 1-5, 15, na 16 (ekishushani S6). Okubaho kwa puroteyini-W kwine akakwate kakuru n’omutindo gwa LH1. TMH zaayo ishatu nizikwatanisibwa n‘enyuguta ngufu, n‘ekicweka kya N aha rubaju rw‘ekicweka ky‘omubiri ekigumire kandi C-terminal aha rubaju rw‘ekicweka ky‘omubiri (ebishushani 1A na 3, A kuhika D). Protein-W neekunda munonga amaizi (ekishushani 3B), kandi TMH2 na TMH3 nizikwatagana na LH1αβ-14 kuhangaho ekicweka ky‘omubiri ekirikuhinguraho (ekishushani 3, B na E kuhika G). Enkoragana y‘omubiri neekira kuba erimu ebisigarira bya Phe, Leu na Val omu kicweka ky‘omubiri ekirikuhinguraho. Ebisigarira ebi nibiteerana n’ebiriisa ebitarikukunda amaizi n’erangi za αβ-14. Ebimwe aha bisigire bya polar nabyo nibihwera omu kukwatanisa, harimu n’omukago gwa haidrojeni ahagati ya W-Thr68 na β-Trp42 aha mutwe gw’ekiina ekigumire (ekishushani 3, F na G). Aha mutwe gw’ekibunu, Gln34 eri haihi n’ekibiina kya keto ekya αβ-14 carotenoids. Okwongyera ahari ekyo, molekyu ya n-dodecyl β-d-maltoside (β-DDM) ekashoboororwa, kandi omukira gwayo ogutarikukunda amaizi gukahika aha nkoragana eri ahagati ya puroteyini-W na αβ-14, kandi omukira gw’amaizi nigubaasa kuba guri omu mubiri. Kandi tukareeba ngu ebicweka bya C-terminal resolution ebya puroteyini W na RCH biri haihi munonga, kwonka tibiri omu nkora y‘okukora enkoragana z‘omutaano (ekishushani 1, A na E). Kwonka nihabaasa kubaho okukoragana omu C-terminal amino acids ezitakashobooroirwe ez’ebirungo ebi bibiri, ekirikubaasa kuha omuringo gw’okuhandiika puroteyini-W omu bwire bw’okuteerana kw’ekicweka kya RC-LH114-W.
(A) Protein-W, erikureebana n’enkoragana na LH1αβ14 omu muringo gwa katuuni, eine omurunga gw’aha rubaju oguri nk’omuti (ogurikutukura), ogurikworekwa omu kicweka ky’ekishushani ky’obushoboorozi bw’amashanyarazi (eitaka ry’enzirugavu eririkureebeka gye n’omutindo gwa kontura gwa 0.13). (B) Puroteni-W erikujwekyerwa ekicweka ky’erangi ekitarikukunda amaizi. Emyanya y’omu itaka n’eya charged neeyorekwa omu rangi ya cyan, emyanya etarikukunda amaizi neeyorekwa omu rangi erikwera, kandi emyanya erimu amaizi g’amaani neeyorekwa omu rangi ya orange. (C na D) Protein-W erikworekwa omu katuuni, oku erikwetoorora ni nk’oku eri omu (A) (C), kandi neeyehindurwa 180° (D). Okurugirira aha mwanya omu kukuratana, ebisigarira ebirikutaanisibwa nibitwara enkora y’erangi y’omuhanganzima, ahu N-terminal eri bururu kandi C-terminal eri erikutukura. (E) Puroteni-W omu kureeba kumwe nk’oku kiri omu (A), kandi ebisigarira aha nkoragana ya puroteyini-W:LH1 nibijwekyerwa emiti erimu obubonero obukwatsire. (F) Protein-W neehindurwa 90° kugyeragyeranisa na (E) na LH1αβ14 omu kujwekyera kwa katuuni, kandi kugyeragyeranisa n‘ebisigarira by‘omugati omu kujwekyera kw‘omubaro. Ebisigarira ebiri ahaiguru kuruga omu beta polypeptide nibihandiikwaho. Cofactor neeyorekwa nk’omuti ogurikuhikaana n’erangi y’ekishushani 1, β-DDM evundiire neeyorekwa omu rangi erikutukura, kandi oxygen neeyorekwa omu rangi erikutukura. (G) Okureeba omu (F) nikuhindurwa 180°, n’ebisigarira ebirikureebwa munonga eby’ekihandiiko kya alpha polypeptide.
Puroteni-W neeza omu mwanya gwa αβ heterodimer (eya 15 omu kishushani 1F), obwo neezibira okukinga kw‘orukoba n‘okuhindura αβ heterodimers ishatu ez‘okubanza. Kikakyeberwa ngu enkokora y‘okutemba erikukirayo ey‘okubanza αβ-1 heterodimer kugyeragyeranisa n‘eya butoosha eya firimu ekaba eri 25° kuhika 29° (ekishushani 1, A na E), eyaabaire ehangirwe okutemba 2° kuhika 8° kwa αβ-1 omu RC A sharp contrast-LH116 naF (ekishushani 1, B). Heterodimers eya kabiri n’eya kashatu nizishendamira ahari 12° kuhika 22° na 5° kuhika 10°, nk’oku biri. Ahabw‘okuremesa kwa steric kwa RC, okushendashenda kwa αβ-1 tikurimu omubaro gwa kabiri gwa αβ (ogurikuhikaana na αβ ya 16 omu kishushani 1F), n‘ahabw‘ekyo nikikora ekicweka ekirikureebwa gye omu mpeta ya LH1 (ekishushani 1, A na E) . Ahabw‘okubura αβ heterodimers ibiri, ezirikuhererukayo n‘okufeerwa BChl ina n‘ebiriisa bibiri, tihaine n‘emwe aha biriisa ebirikukwatanisa n‘ekicweka kya αβ-1 ekihindwirwemu, ekirikureetaho empeta ya LH114-W erimu ebiriisa 13 eby‘okurya by‘ebihingwa n‘eby‘okurya 28 eby‘omubiri. Okutebereza kw‘okushoboorora kw‘omu kyanga kw‘ebicweka bibiri eby‘omubiri omu bicweka bya αβ1 kuhika ahari 7 biri ahansi y‘ebyo eby‘omu kicweka ekisigaireho ekya LH1 loop, ekirikubaasa kworeka obuveera bw‘obuhangwa bw‘ekicweka kya LH1 ekiri haihi n‘omwanya gwa RC QB (ekishushani 4) .
Ebishushani bya RC-LH114-W (A na B) na RC-LH116 (C na D) nibyorekwa kuruga omu kureeba kw‘ahaiguru/aha rubaju kumwe (A na B) (A na C) n‘ahaiguru y‘ekiina ky‘ekishushani 1. (B na D). Ebishumuruzo by’erangi nibyorekwa aha rubaju rwa buryo.
Ekindi kyonka ekiine obumanyiso bw’ekirungo ky’omutaano ekiine omubaro gw’okukyebera 1:14 n’ekirungo ky’ebishaka (Rba.) RC-LH1-PufX dimer (13). Kwonka, puroteyini W na PufX tiziine homology erikureebwa, kandi ziine akakwate kakuru n’enkora zaabo eza LH1. PufX ni TMH emwe erimu ekicweka ky‘omubiri ekirikuhererukayo ekya N-terminal ekirikukwatagana n‘orubaju rw‘omubiri orw‘ekicweka kya RC-H (13) omu mwanya ogurikuhikaana na Rps. omubiri gw’omukazi LH116αβ-16. PufX neekora omuhanda gw‘okuhindura kwa quinone/quinolone ahagati ya RC-LH1 n‘ekirungo kya cytochrome bcl kandi eri omu Rba zoona. ekizibu ky’omunda (13). N’obu omugaati gw’ekigambo kimwe n’ekigambo kimwe guraabe guri omu Rba. Ekicweka kya sphaeroides RC-LH1-PufX dimer kiri omu mwanya gw‘okukwatiraho puroteyini W omu RC-LH114-W, kandi omwanya ogurikureetwaho PufX na puroteyini-W guri omu mwanya ogurikwingana (ekishushani S7A). Empaaka eri omu RC-LH114-W nayo neehikaana n‘omuhanda gw‘okuteekateeka ogwa quinone (8) ogwa Pseudomonas rosea LH1, ogurikukorwaho ebirungo ebitarikushushana na puroteyini W nari PufX (ekishushani S7B). Okwongyera ahari ekyo, omuhanda gwa quinone oguri omu Blc. LH1 eya emerald green erikukorwaho okutaho γ subunit emwe (7) neeshangwa omu mwanya nigwo gumwe (ekishushani S7C). N‘obu kiraabe nikigambwaho puroteyini zitari zimwe na zimwe, okureebeka kw‘emihanda egi eya quinone/quinolol omu mwanya gumwe omu RC-LH1 complex nikireebeka nk‘eky‘okureeberaho ky‘okuhinduka kw‘ebintu ebirikuteerana, ekirikworeka ngu omwanya ogurikureetwaho puroteyini W nigubaasa kukora nk‘omuhanda gwa quinone.
Empaaka eri omu nkora ya LH114-W neebaasisa okutandika kw‘ekicweka ky‘omubiri ekirikugumizamu ahagati y‘omwanya gw‘omunda gw‘ekicweka kya RC-LH114-W n‘omubiri gw‘omubiri (ekishushani 1G), omu mwanya gw‘okugaita emyanya ebiri kurabira omu nkora ya puroteyini nk‘oku kiri omu puroteyini. Ekizibu kya RC-LH116 nikishushana n’ekizibu kya Tch ekikingire. Ekizibu ekirikushushana n‘empimbi (22) (Ekishushani 1H). Ahabw‘okugira ngu okujanjaara kwa quinone kurabira omu membrane nikurahuka kukira okujanjaara kurabira omu muhanda mukye gwa puroteyini, orukoba rwa LH114-W orukingire nirubaasa kwikiriza RC kuhinduka juba kukira orukoba rwa LH116 orukingire, kandi okujanjaara kwa quinone omu RC nikubaasa kuba kukye munonga. Okubaasa kugyezaho yaaba puroteyini W neekwata aha kuhindura kwa quinones kurabira omu RC, tukakora okucondooza kwa cytochrome oxidation aha nkora emwe eya ubiquinone 2 (UQ2) (ekishushani kya UQ10 ey’obuhangwa erimu omukira mukye gwa isoprene) (ekishushani 2E). N‘obu okubaho kw‘ekiriisa kya chelated quinone kuraabe nikuremesa okumanyisa okuhikire okurikureebeka nk‘okwa Michaelis (RC-LH114-W na RC-LH116 nizihikaana na 0.2±0.1μM na 0.5±0.2μM, buri kimwe), omubaro gw‘ahaiguru gwa RC-LH2.RC114-1. 28±5% nizikira ahari RC-LH116 (3.6±0.1 e-RC-1 s-1).
Omu kubanza tukateekateeka ngu puroteyini-W eri omu bicweka nka 10% eby’ekicweka ky’omutaano (16); aha, emihendo y‘okutuura omu butafaari oburikukura omu mushana mukye, omushana gwagati, n‘omushana mwingi ni 15±0.6%, 11±1% na 0.9±0.5, buri kimwe % (ekishushani 2F). Okugyeragyeranisa kw‘omubaro gwa mass spectrometry kukareebeka ngu okwongyeraho histidine tag tikukyendeize aha bwingi bw‘ebiriisa-W kugyeragyeranisa n‘ebika by‘omukibira (P = 0.59), n‘ahabw‘ekyo emiringo egi ti kintu ky‘obuhangwa ekya puroteyini-W ehindwirwemu (ekishushani S10). Kwonka, okutuura oku okukye okwa puroteyini-W omu RC-LH1 complex nikubaasa kureetera RC ezimwe kuhinduka aha sipiidi y‘okurahuka, obwo nizikyendeeza aha kuhindura quinone/quinolone mpora omu RC-LH116 complex. Twareeba ngu omubaro gw‘okutuura omu mushana gw‘ahaiguru tigurikuhikaana n‘ebihandiiko bya transcriptomics ebihererukireyo, ebirikworeka ngu okureebeka kw‘obuzaarwa bwa pufW nikweyongyera ahansi y‘omushana gw‘amaani (ekishushani S11) (23). Entaaniso eri ahagati y‘okuhandiika pufW n‘okuta puroteyini-W omu RC-LH1 complex neebuzabuza kandi neebaasa kworeka okutegyeka okugumire okwa puroteyini.
Omu RC-LH114-W, ebiriisa 6 eby‘omutima (CDL), ebiriisa 7 eby‘omubiri (POPC), ebiriisa 1 eby‘omubiri (POPG) n‘ebiriisa 29 ebya β-DDM nibigabwa kandi bikorwaho eky‘okureeberaho omuriyo 6 CDLs, 24 POPCs, 2 POβDMs na 12. RC-LH116 (ekishushani 5, A na B). Omu nkora ezi zombi, CDL eri haihi kuba aha rubaju rwa cytoplasmic orw‘ekizibu, kandi POPC, POPG na β-DDM nizikira kuba aha rubaju rwa luminal. Ebicweka bibiri by‘amaizi n‘eby‘okushemeza bikataanisibwa omu kicweka kya αβ-1 kuhika αβ-6 eky‘ekicweka kya RC-LH114-W (ekishushani 5A), kandi bitaano bikataanisibwa omu kicweka ekirikugyeragyeranisibwa na RC-LH116 (ekishushani 5B ). Amashaju maingi gakashangwa aha rubaju orundi orw‘ekizibu, okukira munonga CDL, gateeraine ahagati ya RC na αβ-7 kuhika ahari αβ-13 (ekishushani 5, A na B). Ebindi bishaju ebishobooroirwe omu nkora y‘omubiri n‘ebintu ebirikushemeza nibishangwa aheeru y‘empeta ya LH1, kandi enjegyere za acyl ezishobooroirwe gye nizijanjaara ahagati y‘ebicweka bya LH1, ebitebeekanisiibwe omu bwire bukye nka β-DDM omu RC-LH114-W, kandi nibishoboororwa nka β-DDM omu RC A omutaano gwa βPOPC-DHDM na 166. Emyanya erikushushana ey‘okusheeshasheesha n‘ebintu ebirikushemeza omu nkora yaitu neeyoreka ngu n‘emyanya y‘okukwatiraho erikukwata aha mubiri (ekishushani S12A). Emyanya y’ebicweka by’omubiri ebirikushushana omu Tch nayo eine okutaikirizana kurungi. Omucureezi kandi Trv. Omuringo 970 RC-LH1s (ekishushani S12, B kuhika E) (9, 12) n‘ebisigarira ebirikukwatanisa n‘amaizi g‘omubiri eby‘ekibiina ky‘omutwe gw‘amaizi byorekire okurinda kurungi omu nkora y‘okukuratana (ekishushani S13), ekirikworeka ngu Conserved CDL erikukwatanisa na RC (24), emyanya egi eri omu comp- HRCx erikurindwa.
(A na B) RC-LH114-W (A) na RC-LH116 (B) peptides nizijwekyerwa katuuni, kandi erangi nizijwekyerwa emiti, barikukoresa enkora y’erangi eri omu kishushani 1. Ebirungo by’amaizi nibyorekwa omu rangi erikutukura, kandi eby’okushemeza nibyorekwa omu rangi erikutukura. UQ ebohirwe aha myanya ya RC QA na QB eri eya kyenju, kandi UQ erikwetaanisa aha bandi eri eya bururu. (C na D) Emiteekateekyere niyo emwe na (A) na (B), n‘ebishaju bitarimu. (E kuhika G) Okureeba okuhango kwa Q1(E), Q2(F) na Q3(G) kuruga omu RC-LH116, n‘enjegyere z‘aha rubaju ezirikukwatagana. Ebikwato bya haidrojeni nibyorekwa nk’enyuguta ezirikwiragura ezirimu obubonero.
Omuri RC-LH116, RC QA na QB UQ zombi, ezirikwejumba omu kuhingisa kwa electron omu nkora y’okwahukana kwa charge, nizivunda omu myanya yaazo y’okuzikwatanisa. Kwonka omu RC-LH114-W, QB quinone tekashobooroirwe kandi neija kugambwaho omu bwijwire ahaifo. Oyihireho QA na QB quinones, molecules ibiri eza UQ ezirimu chelated (eziri ahagati y‘empeta za RC na LH1) nizigabwa omu nkora ya RC-LH114-W kurugiirira aha guruupu zaabo z‘omutwe ezishobooroirwe gye (eziri omu Q1 na Q2, nk‘oku biri). omwanya). Ekishushani 5C). Ebicweka bibiri bya isoprene nibiheebwa Q1, kandi maapu y‘obuhango neeshoboorora emikira 10 ya isoprene ehikire eya Q2. Omu nkora ya RC-LH116, molekyu ishatu eza chelated UQ10 (Q1 kuhika Q3, ekishushani 5D) zikashoboororwa, kandi molekyu zoona ziine oburemeezi oburikworeka gye omu mukira gwona (ekishushani 5, D kuhika G). Omu nkora ibiri, emyanya y‘ebicweka by‘omutwe gwa quinone ebya Q1 na Q2 eine okutaikirizana kurungi munonga (ekishushani S12F), kandi neekwatagana na RC yonka. Q1 eri aha muryango gw‘ekicweka kya W ekya RC-LH114-W (ekishushani 1G na 5, C, D na E), kandi Q2 eri haihi n‘omwanya gw‘okukwatiraho QB (ekishushani 5, C, D) na F). Ebisigarira bya L-Trp143 na L-Trp269 ebibiikire biri haihi munonga na Q1 na Q2 kandi nibireetaho enkoragana ezirikubaasa kubaho ez‘okuteerana π (ekishushani 5, E na F, n‘ekishushani S12). L-Gln88, 3.0 Å kuruga omu oxygen eri hare eya Q1, neeha omukago gw’amaani ogwa haidrojeni (ekishushani 5E); ekisigaireho eki nikibiikwa omu RC zoona okwihaho enkoragana erikukirayo hare (ekishushani S13). L-Ser91 neehindurwa omu muringo gw‘okurinda Thr omu RC ezindi nyingi (ekishushani S13), ni 3.8 Angstroms kuruga omu methyl oxygen ya Q1, kandi neebaasa kuha enkoragana z‘amaizi ezitaine maani (ekishushani 5E). Q3 neereebeka nk‘etaine nkoragana y‘omutaano, kwonka eri omu kicweka ky‘amaizi ahagati y‘ekicweka kya RC-M n‘ekicweka kya LH1-α 5 kuhika 6 (ekishushani 5, D na G). Q1, Q2 na Q3 nari ebiri haihi ebirimu chelated quinones nabyo bishoboroirwe omu Tch. Omucureezi, Trv. Omuringo gwa 970 na Blc. Omuringo gw‘amaisho (9, 10, 12) nigworeka omwanya gw‘okurinda ogw‘okuhwera quinone ogw‘okukwatiraho omu RC-LH1 complex (ekishushani S12G). UQs itaano ezivundiire omu RC-LH116 nizihikaana gye na 5.8±0.7 ya buri kimwe ekigumire ekirikumanyirwa aha nkora y‘okucondooza ey‘amaizi (HPLC), obwo UQs ishatu ezivunzire omu RC-LH114-W ziri ahansi y‘Omuhendo ogupimirwe ogwa 6.2±0.3 nigworeka ngu hariho uns1.3 (Fig. ekyombeko.
Polypeptides L na M ezitarikushushana buri emwe eine TMH itaano kandi neekora heterodimer erikugaita dimer emwe eya BChl, dimer ibiri eza BChl, monomers ibiri eza bacteriophage (BPh), n’ekyoma kimwe ekitari kya Heme hamwe na molekyu emwe nari ibiri eza UQ10. Kurabira omu kubaho kw‘enkoragana z‘amaizi aha guruupu ya ketoni ey‘aha muheru n‘okuteerana kwayo okumanyirwe omu Rps, ebiriisa bya karotenoidi nibiteebwa omu M-subunit, erikwetwa cis-3,4-dehydroorhodopin. Ebika (25). Ekicweka ky‘aheeru ky‘ekibunu kya RC-H nikihamibwa aha kibunu na TMH emwe. Omuringo gwa RC gwona nigushushana n‘ebicweka bishatu bya RC eby‘ebika ebiine akakwate (nka Rba). ebihimba (PDB ID: 3I4D). Macrocycles za BChl na BPh, omugongo gwa carotenoid n‘ekyoma ekitari kya heme nibikwatagana omu nkora y‘okushoboorora eby‘obuhangwa ebi, nk‘oku ekibiina ky‘omutwe kya UQ10 aha mwanya gwa QA na QB quinone aha RC-LH116 (ekishushani S15).
Okubaho kw‘ebicweka bibiri bya RC ebiine emihendo y‘okutuura omu mwanya gwa QB etari emwe n‘emwe nikiha omugisha musya gw‘okushwijuma empindahinduka z‘omubiri ezitarikuhinduka ezirikuhererukayo n‘okukwatira hamwe kwa QB quinone. Omu nkora ya RC-LH116, QB quinone neeshangwa omu mwanya “oguri haihi” ogubohirwe omu bwijwire (26), kwonka okwahukana kwa RC-LH114-W tikuriho QB quinone. Tihariho QB quinone omu RC-LH114-W, ekirikutangaaza ahabw‘okuba ekizibu eki nikikora, kukira ekizibu kya RC-LH116 ekiine QB quinone erikushoboororwa omu nkora. N’obu empeta ibiri eza LH1 ziraabe nizikora chelate nka quinones mukaaga, itaano nizishoboororwa omu nkora omu mpeta ya RC-LH116 ekingire, obwo eshatu zonka ziine obugarukiro omu nkora omu mpeta ya RC-LH114-W eigukire. Oburemeezi obu obw‘omubiri oburikwongyezibwaho nibubaasa kworeka okugarukaho kw‘ahonaaho kw‘emyanya ya RC-LH114-W QB, okurahuka kw‘okutambura kwa quinone omu kizibu, n‘okwongyera aha kubaasa kw‘okuraba omu nkora ya LH1. Nituteekateeka ngu okubura UQ omu mwanya gwa RC QB ogwa RC-LH114-W nikibaasa kuba nikirugamu ekizibu ekigumire kandi ekirikukorera kimwe, kandi omwanya gwa QB ogwa RC-LH114-W gukaba gukyebeirwe ahonaaho omu kuhinduka kwa UQ Omuringo gw‘omutaano (omuryango gw‘okutaaha omu mwanya gwa QB gukingirwe a the conformation ref.
Hatariho QB, okuhindura okurikuhererukayo okwa L-Phe217 kuza omu mwanya ogutarikuhikaana n‘okukwatira hamwe kwa UQ10, ahabw‘okuba nikwija kureetera okuteerana kw‘omwanya n‘ekicweka ky‘okubanza eky‘isoprene eky‘omukira (ekishushani 6A). Okwongyera ahari ekyo, empindahinduka z‘omubiri ezikuru ezirikureebwa nizireebeka gye, namunonga helix de (helix ngufu omu nkora eri ahagati ya TMH D na E) ahu L-Phe217 erikuhindurwa ekaza omu nsaho y‘okukwatira QB n‘okuhinduka kwa L-Tyr223 (ekishushani 6A ) Kuhenda omukago gwa hydrogen n‘omutindo gw‘okukwatira hamwe ogwa M-5. omwanya (Ekishushani 6B). Helix de neehinduka aha ntandikwa yaayo, Cα ya L-Ser209 neehindurwa 0.33Å, kandi L-Val221Cα neehindurwa 3.52Å. Tihariho mpindahinduka ezirikureebeka omu TMH D na E, ezirikubaasa kuteebwa aha mutwe omu nkora zombi (ekishushani 6A). Nkoku turikumanya, ogu n’omutindo gw’okubanza omu RC y’obuhangwa ogurikukingaho omwanya gwa QB. Okugyeragyeranisa n‘omutindo oguhikire (QB-bound) nikworeka ngu quinone etakakyendeire, empindahinduka y‘omuringo neeyetengyesa kubaasa kugikora kutaaha omu quinone. L-Phe217 neeyehindura kukora enkoragana ya π-stacking n’ekibiina ky’omutwe gwa quinone, kandi helix neehindukira aheeru, ekirikureetera amagufa ga L-Gly222 n’orujegyere rw’aha rubaju rwa L-Tyr223 kukora omukura gw’omukago gwa haidrojeni n’omutindo gw’omukago gwa haidrojeni oguhamire (ekishushani 6, A na C).
(A) Katuuni erikweshushanirizaho eya hologiramu (orujegyere rwa L, orujegyere rwa orange/M, magenta) n‘omutindo gwa apo (enzirugavu), omu bisigire by‘ebikuru birikworekwa omu muringo gw‘okujwekyera nk‘enkoni. UQ10 neejwekyerwa omubaro gwa kyenju. Omuringo ogurimu obutonyezi nigworeka enkoragana ya haidrojeni erikubaho omu nkora yoona. (B na C) Okujwekyera kw’ahaiguru kwa apolipoprotein n’omutindo gw’empeta yoona, okurikworeka oxygen y’omurunga gw’aha rubaju rwa L-Phe217 omu bururu na L-Tyr223 omu rangi erikutukura, nk’oku biri. Ekicweka kya L n’eky’omucungwa; ebicweka bya M na H tibiine rangi. (D na E) Emyanya ya RC QB (D) n‘ebiriisa byona (E) [erangi na (A) nk‘oku biri] na Thermophilus PSII (ekibira, bururu n‘ekirungo kya pulasitiki; PDB ID: 3WU2) Kuteerana (58).
Tikirikuteekateekwaho, n‘obu emiringo etari emwe n‘emwe y‘okubura QB-deficient RCs etaine LH1 eraabe eriho, empindahinduka z‘omubiri ezaareebekire omu kucondooza oku tizikagambirweho enyima. Ebi nibitwariramu omutindo gw’okukyendeeza QB kuruga Blc. (27), Tch. ekicweka ky’omubiri (28) na Rba. sphaeroides (PDB ID: 1OGV) (29), byona biri haihi kushushana n’omutindo gwabyo ogwa QB gwona. Okukyebera gye 3PRC kukashohoza ngu molekyu z‘okushemeza LDAO (Lauryl Dimethyl Amine Oxide) nizikwatanisa aha muryango gw‘omwanya gwa QB, ekirikubaasa kuzibira okugaruka kutebeekanisa omu nkora ekingire. N‘obu LDAO eraabe etarikuvunda omu mwanya gumwe omu 1EYS nari 1OGV, RC ezi nizitebeekanisibwa barikukoresa omubazi gumwe n‘ahabw‘ekyo nizibaasa kurugamu ekintu kimwe. Omubiri gw’ekibiriiti ogwa Rba. Sphaeroides RC erikuteerana n’ekirungo ky’omubiri (PDB ID: 1L9B) nayo neeshusha ngu eine omwanya gwa QB ogukingire. Kwonka omu muringo ogu, ekicweka kya N-terminal ekya RC-M polypeptide (ekirikukoragana n’omwanya gw’okukwatiraho QB kurabira omu H bond y’ebisigarira bya Tyr aha Q helix) nikitwara enkora etari y’obuhangwa, kandi empindahinduka y’enkora ya QB terikugaruka kucondoozibwa (30 ). Ekirikugaruramu amaani n‘okugira ngu titukareebaho okuhinduka kw‘omuringo ogu okwa M polypeptide omu nkora ya RC-LH114-W, okuri haihi kushushana n‘ekicweka kya N-terminal ekya RC-LH116 RC. Kandi kishemerire kumanya ngu bwanyima y‘okumaraho antenna ya LH1 erikwegamira aha detergent, apolipoprotein RCs omu PDB zikashoboororwa, ekyaihireho ebiziba by‘omunda bya quinone n‘amaizi g‘omunda omu mwanya oguri ahagati ya RC n‘omunda y‘ahaiguru y‘empeta ya LH1 eyehinguririize (31, 32). RC neeguma neekora ahabw‘okuba neeguma eine cofactors zoona, okwihaho QB quinone erikuvunda, etarikugumaho munonga kandi erikukira kufeerwa omu bwire bw‘okutebeekanisa (33). Okwongyera ahari ekyo, nikimanyirwa ngu okuihaho LH1 n‘ebishaju by‘obuhangwa ebirikwetoorora kuruga omu RC nibibaasa kugira eki byakora aha mirimo, nk‘okukyendeeza aha myaka y‘amagara g‘embeera ya P+QB-etaanisiibwe eshagama (31, 34, 35). Nahabwekyo, nituteekateeka ngu okubaho kw‘empeta ya LH1 ey‘omu kyanga eyehinguririize RC nikubaasa kuguma n‘omwanya gwa QB “ogukingire”, obwo nikurinda eby‘obuhangwa by‘omu kyanga haihi na QB.
N‘obu apolipoprotein (etariho QB quinone) n‘omutindo gw‘ebintu byona biraabe nibijwekyera ebishushani bibiri byonka eby‘okuhinduka kw‘omwanya gwa QB, beitu kutari omubaro gw‘ebintu ebirikubaho, hariho obumanyiso ngu okukwatira hamwe nikubaasa kukingwaho okuzibira okugaruka kukwatira hamwe na hydroquinone Kuzibira okuzibira kw‘ebicweka by‘omubiri. Enkoragana ya quinolol na quinone haihi n‘omwanya gwa QB ogwa apolipoprotein neebaasa kuba etarikushushana, ekirikureetaho okwangwa kwayo na RC. Kikagambwaho kumara obwire buraingwa ngu empindahinduka z‘omubiri nizikora kihango omu kukwatira hamwe n‘okukyendeeza aha quinones. Obushoboorozi bwa RCs ezikozirwe omu njura kukyendeeza aha quinones bwanyima y‘okumanyiira omwirima nibukyendeera (36); X-ray crystallography neeyoreka ngu okusiisikara oku nikureetwaho QB quinones kukwatwa omu nkora “eri hare” nka 4.5 Å kuruga omu mwanya oguri haihi ogurikukora (26) , 37). Nituteekateeka ngu okuteerana oku okwa distal binding conformation n‘ekishushani ky‘embeera y‘ahagati ahagati ya apolipoprotein n‘omutindo gw‘empeta yoona, ekirikukuratira okukoragana kw‘okubanza na quinone n‘okukingura omwanya gwa QB.
Omuringo gwa II RC ogurikushangwa omu PSII complex ya bacteria ezimwe ezirikurya omushana na cyanobacteria, algae n’ebimera biine okurinda kw’enkora n’emirimo (38). Okuteerana kw‘enkora okurikworekwa omu kishushani 6 (D na E) nikuhamya okushushana ahagati ya PSII RCs n‘omwanya gwa QB gw‘ekicweka kya RC ky‘obukooko. Okugyeragyeranisa oku kumazire obwire buraingwa kuri eky‘okureeberaho ky‘okwega enkora eziine akakwate ka haihi n‘okukwatira hamwe n‘okukyendeeza kwa quinone. Ebihandiiko ebyabandize bikagira ngu empindahinduka z‘omubiri nizigyenda n‘okukyendeeza kwa PSII okwa quinones (39, 40). Nahabwekyo, kuteekateeka aha kurinda RC omu nkora y’okutunguuka, omuringo ogu ogw’okukwatira hamwe ogwabaire gutakareebekaga ira nigubaasa n’okukoresibwa aha mwanya gwa QB ogwa PSII RC omu bimera ebirikurya ebiriisa by’omushana ebirimu oxygen.
Rps ΔpufW (okusazibwamu kwa pufW okutaine kamanyiso) na PufW-His (C-terminal 10x His-eine akamanyiso ka puroteyini-W erikworekwa kuruga omu mwanya gwa pufW ogw‘obuhangwa) ebika. palustris CGA009 ekashoboororwa omu murimo gwaitu ogwabandize (16). Ebika ebi n’omuzaire w’omuringo gw’omukibira ogwa isogenic bikagarurwaho kuruga omu firiiji barikuteera obutafaari bukye ahari PYE (buri 5 g lita -1) (ebibiikire omu LB ahari -80 °C, birimu 50% ( w/v) glycerol) puroteyini, ekirungo ky’ekitumbisa na succinate (%) pla]w [1)v. Empapura zikateebwa omu mwirima ekiro kyona omu bwire bw’obutagatsi bw’omu kishengye omu mbeera ezitarimu n’omwika, reero bwanyima zaamurikwa n’omushana ogurikwera (~50 μmolm-2 s-1) ogwaheebwayo OSRAM 116-W halogen bulbs (RS Components, UK) kumara ebiro 3 kuhika ahari 5 kuhitsya obu ekibunu kimwe kyareebeka. Koloni emwe ekakozesibwa kuteeka miriyoni 10 z’omubazi gwa M22+ (41) oguyongyeirwemu 0.1% (w/v) casamino acids (aha muheru nizimanyirwa nka M22). Obuhangwa bukakurira omu mbeera y’omwika mukye omu mwirima ahari 34°C n’okutengeeta aha 180 rpm kumara eshaaha 48, reero miriyoni 70 z’obuhangwa zikateebwamu omu mbeera niyo emwe kumara eshaaha 24. Obuhangwa bwa semi-aerobic oburimu obuhango bwa miriyoni 1 nibukoresibwa kukyebera miriyoni 30 z‘omubazi gwa M22 omu cupa y‘endabirwamu erikureebeka gye eri aha mutwe gwa miriyoni 30 kandi erikurabwa n‘okugihindura (~50μmolm-2 s-1) kumara eshaaha 48 n‘amaani g‘amashanyarazi agatarimu nshonga Stirring rod. Bwanyima miriyoni 30 z’obuhangwa zikateebwamu lita nka 1 z’obuhangwa omu mbeera niyo emwe, ezaakozesiibwe kukyeberwa lita nka 9 z’obuhangwa oburikwaka aha ~200 μmolm-2 s-1 kumara eshaaha 72. Obutafaari bukasharurwa omu kukyebera aha 7132 RCF kumara edakiika 30, bwagaruka bwateebwa omu ~10 ml ya 20 mM tris-HCl (pH 8.0), kandi bwabiikwa aha -20°C kuhitsya obu bwayetengyesa.
Bwanyima y’okusheenya, yongyera aha butafaari obugarukiremu kusheesheka, yongyeraho ebirungo bimwe eby’ekiriisa kya deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) n’ebirungo bibiri bya Roche holoenzyme puroteyini (Merck, UK) omu butafaari obugarukiremu kusheesheka. Omu kacweka ka 20,000 psi aka Bufaransa (Aminco, USA), obutafaari bukateganisibwa emirundi 8 kuhika ahari 12. Bwanyima y’okuihaho obutafaari obutahendagurikire n’ebisigarira ebitarikubaasa kusheesheka barikubiteeka aha 18,500 RCF kumara edakiika 15 ahari 4°C, omubiri gukaruga omu lysate erimu erangi barikubiteeka aha 113,000 RCF kumara eshaaha 2 ahari 43,000°C. Oteere ekicweka ekirikusheesheka kandi ogarukye oshukyemu omubiri gw’erangi omu miriyoni 100 kuhika 200 za 20 mM tris-HCl (pH 8.0) kandi okore homogenize kuhitsya obu hatariho ebicweka ebirikureebeka. Ekicweka ky’omubiri ekyabaire kihanikire kikateebwa omu 20 mM tris-HCl (pH 8.0) (Anatrace, USA) erimu 2% (w/v) β-DDM kumara eshaaha 1 omu mwirima ahari 4°C n’okugiteeraho mpora. Bwanyima okore centrifuge aha 70°C kubaasa kusheenya 150,000 RCF aha 4°C kumara eshaaha 1 kwihaho ebisigaireho ebitarikusheesheka.
Ekirungo ky‘okushohoza ebiriisa kuruga omu nkora ya ΔpufW kikateebwa aha nkora y‘okuhindura ebiriisa bya DEAE Sepharose eya miriyoni 50 erimu ebiriisa bishatu (CV) eby‘okuteerana [20 mM tris-HCl (pH 8.0) erimu 0.03% (w / v) β-DDM]. Oyoze enyomyo n’ebikwato bibiri ebirikukwatira hamwe CV, reero oyoze enyomyo n’ebikwato bibiri ebirikukwatira hamwe ebirimu 50 mM NaCl. Ekizibu kya RC-LH116 kikakyeberwa n’omubaro gw’omutindo gwa 150 kuhika 300 mM NaCl (omu nkora y’okukwatiraho) ahari 1.75 CV, kandi ekirungo ky’okukwatiraho ekyasigaireho kikakyeberwa n’ekirungo ky’okukwatiraho ekirimu 300 mM NaCl ahari 0.5 CV. Kurundaana ebiri omu nkora y‘okunywanisa ebiri ahagati ya 250 na 1000 nm, okuguma n‘ekicweka ekiine omubaro gw‘okunywanisa (A880/A280) ekirikukira ahari 1 ahari 880 kuhika 280 nm, okikyendeeze emirundi ebiri omu buffer y‘okukwatiraho, kandi ogarukye okozese omuringo nigwo gumwe aha nkora ya DEAE On purification. Dilute obucweka oburimu A880/A280 ratios eri ahaiguru ya 1.7 na A880/A805 ratios eri ahaiguru ya 3.0, okore orurengo rwa kashatu orw’okuhindura ion, kandi ogume obucweka oburimu A880/A280 ratios eri ahaiguru ya 2.2 na A880/A805 ratios eri ahaiguru ya 3.0. Ekicweka eki ekyabaire kishemeziibwe kikateebwa omu nkora kuhika ahari ~2 ml omu kicupuri ky’okukyebera eky’oburemeezi bw’okusharaho (MWCO) (Merck, UK), kandi kyateebwa aha nkora y’okukyebera ey’obuhango bwa Superdex 200 16/600 (GE Healthcare, US) erimu buffer eC m thelun omu 200 m thelun. 1.5 Empapura z'amakuru. Kurundaana ebishushani by‘okunywanisa eby‘ekicweka ky‘okutaho obuhango, kandi ote omutima aha bishushani by‘okunywanisa n‘ebicweka bya A880/A280 aha 2.4 na A880/A805 ahaiguru ya 5.8 kuhika ahari 100 A880, kandi ahonaaho obikorese omu kutebeekanisa nari okubiika ebiri omu cryo-TEM grid Keep at -8.8.
Ekirungo ky‘okushohoza amaizi kuruga omu ruganda rwa PufW-His kikateebwa aha nkora ya HisPrep FF Ni-NTA Sepharose eya miriyoni 20 (20 mM tris-HCl (pH 8.0) erimu NaCl 200 mM na 0.03% (w/w)) omu nkora ya IMca y‘amagara ( GE). v) β-DDM]. Enyomyo ekayozibwa na CV itaano z‘ekirungo kya IMAC, reero bwanyima na CV itaano z‘ekirungo kya IMAC ekirimu 10 mM histidine. Ekicweka ky‘omutaano kikaruga omu nkora n‘ebikwato bitaano bya IMAC ebirimu 100 mM histidine. Ekicweka ekirimu ekirungo kya RC-LH114-W nikiteebwa omu nkora kuhika ahari ~10 ml omu tanka erimu ekicweka ky’okushwijuma Amicon 100,000 MWCO (Merck, UK), kikyebeirwe emirundi 20 n’ekirungo ky’okugikwatanisa, reero kiyongyerwamu miriyoni 25 z’ekirungo kya DEAE fo phaurse kuhika ahari, omu maisho. Oyoze enyomyo n‘ebikwato bina ebirikukwatira hamwe CV, reero oshukye ekizibu aha CV munaana aha nkora y‘okuzibira 0 kuhika 100 mM NaCl (omu kirungo ky‘okukwatira hamwe), kandi CV ina ezisigaireho zirimu ebikwato ebirikukwatira hamwe 100 mM Ebirungo ebisigaireho chlobin A8de20. ahaiguru ya 2.4 n‘omubaro gwa A880/A805 oguri ahaiguru ya 4.6 ebicweka bikaba biteirwe omu nkora kuhika ahari ~2 ml omu kicweka ky‘okushwijuma eky‘omugati ekya Amicon 100,000 MWCO, kandi byaijura 1.5 CV IMAC omu maisho Buffer erikwingana Superdex 200 ahaiguru y‘ekicupuri ky‘okusheeshasheesha omu 16/600. 1.5 Empapura z'amakuru. Kurundaana ebishushani by‘okunywanisa eby‘ebicweka by‘obuhango-okutaho kandi ote omutima aha bishushani by‘okunywanisa n‘ebicweka bya A880/A280 aha 2.1 na A880/A805 ahaiguru ya 4.6 kuhika ahari 100 A880, ebirikukoresibwa ahonaaho omu kutebeekanisa TEM grid erimu obufuki nari ebibiikwa aha -80 C.
Furiizi y‘okuta omu maizi eya Leica EM GP ekakoresibwa kutebeekanisa ebipande bya TEM eby‘obutagatsi bukye. Ekizibu eki kikakyeberwa omu IMAC buffer kuhika ahari A880 ya 50, reero 5μl zikateebwa aha nkora y’omuringa eya QUANTIFOIL 1.2/1.3 eyaabaire erimu kaboni (Agar Scientific, UK) eyaabaire eherize kusheesheka. Oteere gridi aha 20°C na 60% y’obutagatsi oburikuhikaana n’obwire kumara 30 s, reero ogifukye eyomire kumara s 3, reero ogizime omu maizi ga ethane ahari -176°C.
Ebihandiiko by’ekicweka kya RC-LH114-W bikahandiikwa aha eBIC (Ekitongore ky’okucondooza eby’amagara) (Ekitongore ky’omushana gw’eby’amagara ekya Bungyereza) n’ekikwato kya Titan Krios, ekirikukorera aha amaani g’okutunguura amaani ga 300kV, n’okutunguuka kw’eiziina kwa Choose gap 130,000 of-2010. Gatan 968 GIF Quantum erimu ekikwato ky’okumanyisa enshonga ya K2 kikakozesibwa kuhandiika ebishushani omu muringo gw’okubara kubaasa kushorooza amakuru. Obuhango bwa pixel obugyeziibwe ni 1.048Å, kandi omubaro gw’eidaara ni 3.83 e-Å-2s-1. Akateerana firimu omu bwire bwa sekondi 11 kandi yaagibaganisamu ebicweka 40. Koresa omwanya ogurimu kaboni kugaruka kuta omutima aha maikorosikopu, reero oteerane firimu ishatu ahari buri kiina. Omu bwijwire, firimu 3130 zikashoroozibwa, n‘emihendo y‘okuta omutima aha nshonga eri ahagati ya -1 na -3μm.
Ebihandiiko by’ekicweka kya RC-LH116 bikashoroozibwa barikukoresa ekikwato nikyo kimwe omu kitongore ky’okukyebera eby’obuhangwa ekya Asterbury (Yunivasite ya Leeds, Bungyereza). Ebihandiiko bikashangwa omu muringo gw’okubara n’okutunguuka kwa 130 k, kandi obuhango bwa pixel bukagyeragyeranisibwa kuhika ahari 1.065 Å n’omubaro gwa 4.6 e-Å-2s-1. Firimu egi ekakwatwa omu bwire bwa sekondi 12 kandi ekabaganisibwamu ebicweka 48. Omu bwijwire, firimu 3359 zikatorotoorwaho, n‘emihendo y‘okuta omutima aha nshonga eri ahagati ya -1 na -3μm.
Okukora aha makuru goona nikukorwa omu nkora ya Relion 3.0 (42). Koresa Motioncorr 2 (43) kushoboorora okutambura kw‘omuti orikukoresa oburemeezi bwa dozi, reero okozese CTFFIND 4.1 (44) kumanyisa CTF (omurimo gw‘okuhingisa okutaana) paramita. Ebishushani by‘omutaano eby‘okureeberaho bwanyima y‘emitendekyere egi ey‘okubanza y‘okukora nibyorekwa omu kishushani 2. S16. Enkora y‘okutooranamu ey‘okwekorera neekorwaho okutoorana omu ngaro nka pixels nka 250 z‘ebicweka 1000 omu fureemu ya pixels 250 kandi hatariho kushoboorora okushoroora kw‘ebicweka bibiri (2D), obwo nikwanga okushoroora okwo okurikuhikiiriza obukooko bw‘eky‘okureeberaho nari okutaine mitwarize erikureebwa. Bwanyima, okutoorana kw’okwekorera kukakorwa aha bishushani byona ebikye, kandi RC-LH114-W ekaba eri obucweka 849,359, kandi RC-LH116 complex ekaba eri obucweka 476,547. Obucweka bwona obutoorainwe bubaire nibukora emirundi ebiri y‘okushoroora 2D okutari kw‘okureeberaho, kandi bwanyima ya buri rugendo, obucweka oburikuhikaana n‘omwanya gwa kaboni, okushiisha kw‘eky‘okureeberaho, obutagira bishushani ebirikureebwa nari obucweka oburikuteerana munonga nibwangwa, ekirikureetaho okushoroora 772,033 (90.9%) na 359.D %) of Parles are357 ( )d . RC-LH114-W na RC-LH116 nk’oku biri. Omuringo gw‘okubanza ogw‘okureeberaho ogwa 3D gukakorwa barikukoresa omuringo gw‘okushuuma kw‘omutindo gwa stochastic gradient. Okukozesa eky‘okureeberaho ky‘okubanza nk‘eky‘okureeberaho, obucweka obutoorainwe nibubaganisibwamu ebicweka bina omu 3D. Okwejunisa eky‘okureeberaho omu kicweka eki nk‘eky‘okureeberaho, kora okushemeza kwa 3D aha bicweka ebiri omu kicweka ekihango, reero okozese ekicweka ky‘okubanza ekya 15Å low-pass filter kukingaho omwanya gw‘okushohoza, yongyeraho pixels 6 z‘empimbi ezigumire, kandi bwanyima y‘okukora pixels kushoboorora Gatan K2 transfer function the top Mo. Aha RC-LH114-W dataset, omuringo ogu ogw‘okubanza gukahindurwa barikwihaho oburemeezi bw‘amaani aha rubaju rw‘ekijwaro ky‘okukinga amaisho (obutakwatagana n‘oburemeezi bw‘amaani obugumire omu UCSF Chimera). Ebishushani ebirugiremu (okushoboorora kwa RC-LH114-W na RC-LH116 ni 3.91 na 4.16 Å, buri kimwe) nibikoresibwa nk‘eky‘okureeberaho ky‘omuringo gwa kabiri gw‘okushoroora 3D. Obucweka oburikukoresibwa nibuteeranirwa omu guruupu y‘okubanza eya 3D kandi tiburimu kakwate k‘amaani n‘omutaahi. Okuteerana nari okubura ebishushani by‘enkora ebirikureebwa. Bwanyima y‘omuringo gwa kabiri gw‘okushoroora 3D, ekicweka ekiine obushoboorozi bw‘ahaiguru kikatooranwa [Aha RC-LH114-W, ekicweka kimwe n‘ebicweka 377,703 (44.5%), ahari RC-LH116, hariho ebicweka bibiri byonka, byona hamwe biri 260,752 obucweka W (%) the aligne. bwanyima y’okubanza kuhindura n’entaaniso nkye]. Ebicweka ebitoorainwe nibigaruka bishohozibwa omu kabokisi ka pixel 400 kandi bishemezibwa omu muringo gwa 3D refining. Ekijwaro ky‘okukinga amaisho nikikorwa barikukoresa ekicweka ky‘okubanza eky‘okushwijuma 15Å eky‘okuhingura ahansi, okugaziha kwa maapu ya pixel 3 n‘ekipande ky‘okukinga ekirikworoba kya pixel 3. Okukozesa okushemeza CTF ahari buri kicweka, okutereeza okutambura kwa buri kicweka n‘omuringo gwa kabiri gw‘okushemeza CTF ahari buri kicweka, okushemeza kwa 3D, okukinga ebiriisa n‘okukora bwanyima y‘okukora nibikorwa bwanyima ya buri ntambwe kwongyera kushemeza ekirugiremu. Okukozesa omuhendo gw‘okusharaho ogwa FSC (Fourier Shell Correlation Coefficient) ogwa 0.143, okushoboorora kw‘eby‘okureeberaho by‘aha muheru ebya RC-LH114-W na RC-LH116 ni 2.65 na 2.80Å, buri kimwe. FSC curve y‘omuringo gw‘aha muheru neeyorekwa omu kishushani 2. S17.
Okukuratana kwa puroteyini kwona nikuheebwa kuruga omu UniProtKB: LH1-β (PufB; ID ya UniProt: Q6N9L5); LH1-α (PufA; Ekirikumanyisa ekintu kimwe: Q6N9L4); RC-L (PufL; endagiriro y’ekicweka kimwe: O83005); RC-M (PufM; endagiriro y’ekicweka kimwe: A0A4Z7); RC-H (PuhA; endagiriro y’ekicweka kimwe: A0A4Z9); Ebirungo-W (PufW; endagiriro ya UniProt: Q6N1K3). SWISS-MODEL (45) ekakozesibwa kwombeka omuringo gw’okushushana ogwa RC, ogurimu okukuratana kwa puroteyini za RC-L, RC-M na RC-H n’omutindo gw’ekibiriiti kya Rba. sphaeroides RC ekakoresibwa nk‘eky‘okureeberaho (PDB ID: 5LSE) (46). Koresa ekikwato kya “maapu ehikire” omu UCSF Chimera kuhikaana n‘eky‘okureeberaho ekikozirwe na maapu (47), kutunguura omutindo gwa puroteyini, n‘ekirikureetaho [4×BChl a (eiziina ry‘ebisigarira by‘omu ibiikiro ry‘ebitabo = BCL), 2×BPh a (BPH), omuringo gumwe nari ebiri gwa UQ10 (U10), gumwe (ieFme) . 3,4-dihayidroheksakarbonilikolini (QAK)] kozesa Coot (48) kwongyeraho. Ahabw‘okugira ngu QAK teriho omu ibiikiro ry‘ebitabo rya monomer, ekateebwaho paramita barikukoresa ekikwato kya eLBOW omu PHENIX (49).
Ekindi, ekicweka kya LH1 kikakorwa. Omu kubanza, ekikwato ky‘okwombeka eky‘okwekorera omu PHENIX (49) kikakoresibwa kwombeka ekicweka ky‘omubaro gwa LH1 omu muringo gw‘okwekorera barikukoresa maapu n‘omubaro gw‘ebiriisa bya LH1-α na LH1-β nk‘ebirikutaaha. Toorana ekicweka kya LH1 ekihikire munonga, okiihemu kandi okite omu Coot, omu ngaro yongyeraho omukuratana ogutarimu, kandi omu ngaro oshemeze omubumbi weena otakayongyeireho BCls ibiri a (BCL) na spirilloxanthin (CRT) [kurugiirira ahari Rps eziine akakwate Oburemeezi bw‘ebiri omu LH1 complex n‘ebiri omu carotenoid ebimanyirwe. Ebika (17)]. Kopa ekicweka kya LH1 ekihikire, kandi okozese UCSF Chimera “Ekikwato kya maapu y‘okutaho” kukwaata omu mwanya oguri haihi ogutari gw‘eky‘okureeberaho gw‘obuhango bwa LH1, reero okishemeze omu Coot; garukamu enkora egi kuhitsya obu ebicweka bya LH1 byona biraabe byakozirweho eky‘okureeberaho. Aha nkora ya RC-LH114-W, okushohoza oburemeezi obutagabirwe omu Coot, puroteyini neebaganisibwamu kuruga omu bicweka ebisigaireho ebitari puroteyini omu maapu ya USCF Chimera kandi ekikwato kya Autobuild nikikoresibwa kutaho eky‘okureeberaho ky‘okubanza, n‘ebicweka ebisigaireho (puroteyini-W) Modeling. Omuri FENISI (49). Yongyera aha nkora yoona erikubura aha nkora erikuruga omu Coot (48), reero oshemeze omu ngaro ekicweka kyona. Oburemeezi obusigaireho obutagabirwe nibuhikaana n‘okuteerana kw‘ebishaju (PDB ID y‘ekitabo ky‘ebitabo bya CDL = CDL, POPC = 6PL na POPG = PGT), ekirungo kya β-DDM (LMT) n‘ebicweka bya UQ10 (U10). Koresa PHENIX optimization (49) n‘okutunguura omu ngaro omu Coot (48) kuhikiiriza eky‘okureeberaho ky‘okubanza ekihikire kuhitsya obu emibaro y‘eky‘okureeberaho n‘omutindo gw‘okureeba ogw‘okuhikaana gutarikubaasa kwongyera kutunguuka. Eky‘aha muheru, kozesa LocScale (50) kushemeza maapu y‘ekyanga, reero okore enzindo ezindi nyingi z‘okugyeragyeranisa obuhango obutagabirwe n‘okutunguura kw‘okwekorera n‘okw‘omu ngaro.
Peptides, cofactors n‘ebindi bihimba n‘ebindi bihingwa ebiine amaizi n‘ebihimba ebiine amaizi (quinones) ebiteirwe omu nkora yaabyo nibyorekwa omu bishushani 1 na 2. S18 kuhika S23. Amakuru g‘embariirira g‘omuringo gw‘aha muheru nigorekwa omu kipande S1.
Okwihaho kyaba kitarikworekwa, ebishushani by’okunywanisa UV/Vis/NIR bikatorotoorwa aha kikwato ky’okupima ebishushani ekya Cary60 (Agilent, USA) aha bwire bwa 1 nm kuruga ahari 250 nm kuhika ahari 1000 nm n’obwire bw’okuteerana bwa 0.1s.
Reeba sampo omu quartz cuvette erimu omuhanda gwa mm 2 kuhika ahari A880 ya 1, kandi oteerane ebiri omu nkora y’okunywa ebiri ahagati ya 400 na 1000 nm. Ebishushani by’erangi ebirikwetoorora bikatorotoorwaho aha kipimo kya Jasco 810 (Jasco, Japan) aha bwire bwa 1 nm ahagati ya 400 nm na 950 nm aha sipiidi y’okukyebera 20 nm min-1.
Omubaro gw‘okuhwaho kwa molar nigumanyirwa aha kukyendeeza aha nkora y‘omubiri (core complex) kuhika ahari A880 erikwingana 50. Kukyendeeza aha bwingi bwa 10μl omu 990μl y‘ekirungo ky‘okuzibira nari methanol, kandi oteerane ebiriisa by‘okunywa ahonaaho kukyendeeza aha kusheeshasheesha kwa BChl. Ebirimu BChl omuri buri sampo ya methanol bikabarwa n’omubaro gw’okuhwaho ahari 771 nm ogwa 54.8 mM-1 cm-1, kandi omubaro gw’okuhwaho gukamanyirwa (51). Gabisa omubaro gwa BChl ogupimirwe na 32 (RC-LH114-W) nari 36 (RC-LH116) kubaasa kumanya omubaro gw‘ebintu ebigumire eby‘omusingi, ogurikukoresibwa bwanyima kumanya omubaro gw‘okunywanisa gw‘eky‘okureeberaho kimwe ekirundainwe omu buffer Extinction coefficient. tibikabugana. Ebipimo bishatu ebyagarukiremu bikakorwa ahari buri sampo, kandi okunywanisa kwa BChl Qy okurikukirayo kukakoresibwa omu kubara. Omubaro gw’okuhwaho kwa RC-LH114-W ogupimirwe ahari 878 nm ni 3280±140 mM-1 cm-1, kandi omubaro gw’okuhwaho kwa RC-LH116 ogupimirwe ahari 880 nm ni 3800±30 mM-1 cm-1.
UQ10 ekakyeberwa kurugiirira aha nkora eri omu (52). Omu bugufu, okucondooza okurikugaruka enyima (RP-HPLC) kukakorwa barikukoresa enkora ya Agilent 1200 HPLC. Okusheeshasheesha nk‘ebicweka 0.02 nmol bya RC-LH116 nari RC-LH114-W omu 50μl ya 50:50 methanol:chloroform erimu 0.02% (w/v) y‘ekyoma ky‘okusheeshasheesha, kandi oshukyemu omubazi gw‘okusheeshasheesha ogwabaire gutebeekanisiibwe kare omu m4. 40°C omu nkora ya HPLC (80:20 omubazi gw’okukyebera:2-omubazi gw’okukyebera) aha nkora ya ×25 cm. Kora okukyebera kw‘isocratic omu nkora ya HPLC kureeba oku okunywa kw‘amaizi kuri 275 nm (UQ10), 450 nm (ekiriisa ky‘omubiri) na 780 nm (BChl) kumara eshaaha 1. Enshonga y’ahaiguru omu 275 nm chromatogram aha dakiika 25.5 ekaba erimu, eyaabaire etarimu ebindi bihingwa ebirikureebeka. Ekicweka ekirimu nikikoresibwa kubara omubaro gwa molar ogwa UQ10 ogwashohoziibwe orikugyendera aha nkora y‘okugyezesa erikubarwa kuruga omu kuteekaho emitindo eshemeire kuruga ahari 0 kuhika ahari 5.8 nmol (ekishushani S14). Buri sampo ekakyeberwa omu kugarukamu kashatu, kandi enshobi eyaagambirweho neehikaana na SD y‘omubaro gw‘ahaiguru.
Omubazi ogurimu ekirungo kya RC-LH1 ekiine okunywanisa kwa Qy okurikukirayo okwa 0.1 gukatebeekanisibwa n‘omutima gw‘embaraasi ogukyebeirwe 30 μM c2 (Merck, UK) na 0 kuhika 50 μMUQ2 (Merck, UK). Sampuli ishatu eza miriyoni 1 zikatebeekanisibwa ahari buri nkora ya UQ2 kandi zaateebwa omu mwirima ekiro kyona ahari 4°C kureeba ngu zaamanyiira kimwe omwirima zitakapimirwe. Omubazi ogu gukateebwa omu OLIS RSM1000 modular spectrophotometer erimu ekikwato ky’okuteeraho 300 nm/500 line grating, 1.24 mm okutaaha, 0.12 mm ahagati na 0.6 mm okutaaha. Ekicweka ky‘okushwijuma ekirikuhinguraho eky‘oburaingwa bwa 600 nm nikiteebwa aha muryango gw‘ekishushani ky‘eky‘okureeberaho n‘ekishushani ky‘okureeberaho eky‘okukanyisa ebishushani kubaasa kubinga omushana gw‘okusiimura. Okunywanisa kukakyeberwa ahari 550 nm n’obwire bw’okuteerana bwa 0.15 s. Omushana gw’okusiimura niguruga omu 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) kurabira omu nkora y’okureeba ebiri omu nkora aha 90% kurabira omu nkora y’okutegyeka DC2200 (Thorlabs Ltd., UK) kandi niguruga omu nshonga y’omushana aha nkora ya90. Omuti gw’okupima niguhakanisa endabirwamu kugaruraho omushana gwona ogwabaire gutarikukwatwaho sampo omu kubanza. Reeba okunywanisa 10 s hatakabaireho omushana gwa 50 s. Bwanyima okunywanisa kukagaruka kwakyeberwa kumara 60 s omu mwirima kureeba oku quinolol erikweyongyera kukyendeeza cytochrome c23 + (reeba ekishushani S8 ahabw‘ebihandiiko ebitahikire).
Ebihandiiko bikakorwaho barikuhikaana n‘omubaro gw‘okubanza ogw‘omuringo gumwe omu bwire bwa 0.5 kuhika 10 s (kurugiirira aha nkora ya UQ2) n‘okugyeragyeranisa emihendo y‘eby‘okureeberaho byona bishatu ahari buri nkora ya UQ2. Ekicweka kya RC-LH1 ekyabariirwe omu nkora y’okuhwaho ekirikushushana kikakozesibwa kuhindura omubaro omu nkora y’okureetaho okuhinduka, ekyateirwe omu Origin Pro 2019 (OriginLab, USA), kandi kyahikaana n’omuringo gwa Michaelis-Menten kubaasa kumanya emihendo ya Km na Kcat erikureebeka.
Aha bipimo by‘okunywanisa eby‘obwire bukye, sampo ya RC-LH1 ekakyeberwa kuhika ahari ~2μM omu IMAC buffer erimu 50 mM y‘okunywanisa (Merck, USA) na 0.4 mM y‘okunywanisa (Merck, USA). Ascorbic acid neekoresibwa nk‘omuhi w‘ekiriisa ky‘okuhayo, kandi tert-butaclofen neekoresibwa nk‘omuhi w‘obuhwezi bwa QB kureeba ngu omuhi w‘obuhwezi mukuru wa RC yaaguma naakyendeera (nikwo kugira ngu, ataine photooxidized) omu nkora yoona ey‘okupima. Enkumi miriyoni 3 z‘eky‘okureeberaho niziteebwa omu nkora erikwetoorora (nk‘oburaingwa bwa mita 0.1 omu bugazi, 350 RPM) n‘oburaingwa bw‘omuhanda gw‘okureeba ogwa mm 2 kureeba ngu eky‘okureeberaho omu muhanda gwa laser kiine obwire burikumara bw‘okuhindura omu mwirima ahagati y‘empumuro z‘okusiimura. Koresa ~100-fs ebiri omu nkora ya laser kutunguura enkora ya laser ya Ti: Sapphire (Spectra Physics, USA) kushemeza sampo aha 880 nm aha rurengo rw‘okugarukamu rwa 1 kHz (20 nJ ahari NIR nari 100 nJ ahari Vis). Otakashorooize amakuru, banza oteho sampo omu mushana gw‘okusiimura kumara edakiika nka 30. Okwemanyiza nikwija kureetera QA kutakora (nikibaasa kukyendeeza QA omurundi gumwe nari ebiri). Kwonka manya ngu enkora egi neebaasa kugaruka enyima ahabw‘okuba bwanyima y‘obwire buraingwa bw‘okumanyiira omwirima, RC neija kugaruka mpora mpora omu mirimo ya QA. Ekipimo kya Helios (Ultrafast Systems, USA) kikakozesibwa kupima ebiri omu bwire bukye n’obwire bw’okukyerererwa bwa -10 kuhika 7000 ps . Koresa puroguraamu ya Surface Xplorer (Ultrafast Systems, USA) kushoroora ebihandiiko ebiri omu guruupu, reero obigaite hamwe kandi obikorese omu muringo gumwe. Koresa puroguraamu ya CarpetView (Light Conversion Ltd., Lithuania) kukoresa ebihandiiko ebiteeraine kutunga ebishushani by‘entaaniso ebiine akakwate n‘okuvunda, nari kozesa omurimo ogurikutaho ebishushani bingi n‘okugarukamu kw‘ekikwato kuhikaana n‘entunguuka y‘ebishushani by‘oburaingwa bw‘omuyaga gumwe omu Origin, USALa (Origin).
Nkoku kyagambirweho ahaiguru (53), firimu y‘okukora eby‘okurya erimu LH1 complex etaine RC n‘antenna ya LH2 ey‘aheeru ekatebeekanisibwa. Ekibunu kikaba kikyebeirwe omu 20 mM tris (pH 8.0) reero kyateebwa omu cuvette ya quartz erimu omuhanda gw’okureeba ogwa mm 2. Laser pulse ya 30nJ ekakoresibwa kushemeza sampo ahari 540 nm n’obwire bw’okukyerererwa bwa -10 kuhika 7000 ps. Okukora aha nkora y‘okuhandiika nk‘oku kyashoboororwa ahari Rps. pal ekyokureeberaho.
Ekicweka eki kikakyeberwa omu kukyebera aha 150,000 RCF kumara eshaaha 2 ahari 4°C, reero okunywa kwakyo ahari 880 nm kukagaruka kwateebwa omu 20 mM tris-HCl (pH 8.0) na 200 mM NaCl. Okusheeshasheesha omubiri orikuguteera mpora mpora omu 2% (w/v) β-DDM kumara eshaaha 1 omu mwirima ahari 4°C. Eky’okureeberaho kikakyeberwa omu 100 mM y’ekiriisa kya puroteyini (pH 8.0) (TEAB; Merck, UK) kuhika aha puroteyini y’ekiriisa kya 2.5 mg ml-1 (okucondooza kwa Bio-Rad). Okwongyera kukoraho kukakorwa kuruga omu nkora eyaabaire eshohoziibwe enyima (54), kutandika n‘okukyendeeza puroteyini 50 μg omu mubaro 50 μl TEAB erimu 1% (w/v) sodium laurate (Merck, UK). Bwanyima y’okukyebera kumara 60 s, kikakyendeezibwa n’okukyebera 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) aha 37°C kumara edakiika 30. Aha S-alkylation, teera sampo n’ekiriisa kya 10 mM (Merck, UK) kandi ogyongyeremu kuruga omu 200 mM isopropanol stock solution kumara edakiika 10 aha butagasi bw’omu kishengye. Okugabura eby’okurya omu mubiri kukakorwa barikuteekamu 2 μg y’omutaano gwa trypsin/endoproteinase Lys-C (Promega UK) kandi bakabiteeka aha 37°C kumara eshaaha 3. Ekirungo kya laurate kikaihwaho omu kugaita 50 μl y’ekiriisa kya ethyl asetate na 10 μl 10% (v/v) LC ey’omutindo gwa trifluoroasetic asidi (TFA; Thermo Fisher Scientific, UK) kandi bakagiteera omu nkungu kumara 60 s. Okwahukana kw‘ebicweka kukatunguuka ahabw‘okukyebera aha 15,700 RCF kumara edakiika 5. Okurugirira aha nkora y’abakozi, enyomyo ya C18 spin column (Thermo Fisher Scientific, UK) ekakoresibwa kusheeshasheesha n’obwegyendesereza n’okushohoza omunyugunyu omu kicweka kya hansi ekirimu ekirungo kya peptide. Bwanyima y’okuyomesa omu nkora y’okukyebera omu mwanya ogutarimu kintu kyona, eky’okureeberaho kikashohozibwa omu 0.5% TFA na 3% ya acetonitrile, kandi 500 ng zikakyeberwa omu nkora ya nanoflow RP chromatography hamwe n’okukyebera kw’obuhango bw’ebintu barikukoresa enkora y’okucondooza eyaabaire eshobooroirwe omu bwijwire enyima.
Kozesa MaxQuant v.1.5.3.30 (56) ahabw‘okumanyisa puroteyini n‘okupima obuhango bw‘okusherura Rps. ebiri omu nkora y‘okuhandiika ebiriisa by‘omubiri (www.uniprot.org/proteomes/UP000001426). Ebihandiiko by’okucondooza aha nkora ya proteomics biteirwe omu mukago gwa ProteomeXchange kurabira omu kitongore ky’abanywani ba PRIDE (http://proteomecentral.proteomexchange.org) ahansi y’ekimanyiso ky’ebihandiiko PXD020402.
Ahabw’okushwijuma RPLC hamwe n’okukyebera obuhango bw’amashanyarazi, ekicweka kya RC-LH1 kikatebeekanisibwa kuruga omu Rps z’omuringo gw’omukibira. Okukozesa omuringo ogwabandize kushohozibwa (16), omubaro gw’ebiriisa ebyakozirwe omu butafaari bwa palustris gukaba guri 2 mg ml-1 omu Hepes 20 mM (pH 7.8), NaCl 100 mM na 0.03% (w/v) β- (okucondooza kwa Bio-Rad) ) DDM. Okurugirira aha nkora y’omukozi, kozesa ekikwato ky’okushemeza 2D (GE Healthcare, USA) okushohoza puroteyini 10 μg omu muringo gw’okusheenya, kandi oshukyeere omu 20 μl 60% (v / v) asidi ya formiki (FA), 20% (v / v) Acetonitrile/ 20% (2)vter (20%). Maikorolita itaano zikakyeberwa RPLC (Dionex RSLC) hamwe n‘okupima obuhango bw‘amaizi (Maxis UHR-TOF, Bruker). Koresa enyomyo ya MabPac 1.2×100 mm (Thermo Fisher Scientific, UK) ahabw‘okwahukana aha 60°C na 100μlmin -1, n‘okukyendeeza aha 85% (v / v) ekirungo A [0.1% (v / v) FA na 0.02% (V/v) ekirungo B5/v a. [0.1%(v/v) FA na 0.02%(v/v) omu 90%(v/v) asetonitrile TFA] Okwejunisa enshonga y‘okufuuhirira kw‘amashanyarazi ey‘omutindo n‘ebicweka by‘okubanza kumara edakiika ezirikurenga 60, ekipimo ky‘oburemeezi nikitunga 100 kuhika 2750 m/z (omubaro gw‘oburemeezi n‘okushangwa). Ahabw‘obuhwezi bw‘ekikwato kya FindPept (https://web.expasy.org/findpept/), maapu y‘eby‘obugaiga by‘omubiri kuhika aha bicweka by‘ekicweka ky‘ekizibu.
Obutafaari bukakuzibwa kumara eshaaha 72 ahansi y’omushana gwa miriyoni 100 NF-ahansi (10μMm-2 s-1), ahagati (30μMm-2 s-1) nari ahaiguru (300μMm-2 s-1). M22 omubazi (omubazi gwa M22 ogu ammonium sulfate erikurekwaho kandi sodium succinate erikuhindurwa sodium acetate) omu cupa ya miriyoni 100 erimu ekikwato ky‘okuteeraho (23). Omu nkora itaano eza 30-s, obubare bw’endabirwamu bwa 0.1 micron bukateebwa aha nkora y’obuhango bwa 1:1 kubaasa kusheenya obutafaari kandi bukafukiirwa aha ice kumara edakiika 5. Ebintu ebitarikubaasa kusheesheka, obutafaari obutahendagurikire n’ebirungo by’endabirwamu bikaihwaho omu kukyebera aha 16,000 RCF kumara edakiika 10 omu nkora y’okukyebera ebiri aha mutwe gw’entebe. Ekicweka ky’omubiri eki kikataanisibwa omu nkora ya Ti 70.1 erimu RCF 100,000 omu 20 mM tris-HCl (pH 8.0) erimu 40/15% (w/w) y’omubiri gw’omubiri kumara eshaaha 10.
Nkoku kyashoboororwa omu murimo gwaitu ogwahingwire, okucondooza aha nkora y‘okurwanisa endwara ya His tag aha PufW (16). Omu bugufu, ekicweka ky‘omubiri ekishemeziibwe (11.8 nM) nari ekicweka ky‘omubiri ekirimu omubaro nigwo gumwe gwa RC (ekirikumanyirwa aha kuhindura ebiri omu nkora (oxidation) okukyendeeza aha spectrum y‘entaaniso n‘okuhikaana n‘omugugu aha gel erimu erangi) omu 2x SDS loading buffer (Merck, UK) erikukyeberwa emirundi ebiri. Ebirungo ebi bikataanisibwa aha nkora y’okushushanisa 12% bis-tris NuPage gel (Thermo Fisher Scientific, Bungyereza). Gel ekateebwaho erangi ya Coomassie Brilliant Bururu (Bio-Rad, UK) kubaasa kutaho n’okureeba omu maisho ekicweka kya RC-L. Puroteni eri aha gel ya kabiri ekatwarwa omu nkora y‘okukyebera obutafaari bw‘omubiri oburikurwanisa endwara. Omubiri gwa PVDF gukazibirwa omu 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 na 5% (w / v) amate agatarimu nshaho, reero bwanyima yaateebwa omu nshaho y‘okurwanisa His ey‘okubanza (omu buffer mH0p 7.6), NaCl 150 mM na 0.05% (v/v) Tween-20] omu 1:1000 A190-114A, omu kitongore ky’okukyebera ekya Bethyl, USA) kumara eshaaha 4. Bwanyima y‘okwozya emirundi 3 kumara edakiika 5 omu nkora y‘okurwanisa endwara, omubiri gukagambwaho n‘enkora y‘okurwanisa endwara ya horseradish peroxidase (Sigma-Aldrich, UK) ey‘okurwanisa endwara y‘omubiri (ekaba erimu 1:10,000 omu nkora y‘okurwanisa endwara) WInkubate kubaasa kuha orusa kukyebera (edakiika 5 bwanyima y‘okwozya emirundi 3 u omu nkora y‘okurwanisa endwara E ESTA0). (Cyanagen, Italy) hamwe na Amersham Imager 600 (Eby’amagara bya GE, Bungyereza).
Omu kuteera ekishushani ky‘okugabwa kw‘amaani ga buri gel erimu erangi nari omuhanda gw‘okukyebera obutafaari bw‘omubiri, okuta omu nkora omwanya oguri ahansi y‘enshonga n‘okubara omubaro gw‘amaani ga RC-L (gel erimu erangi) na Protein-W (immunoassay), omu ImageJ (57) Kora aha kishushani. Ebipimo ebi bikahindurwa byahinduka ebipimo bya molar barikuteekateeka ngu omubaro gwa RC-L na puroteyini-W omu sampo ya RC-LH114-W eshemeire gukaba guri 1:1 kandi bakagaruza busya ebihandiiko byona kurugiirira ahari ekyo.
Okumanya ebihandiiko by’okwongyeraho eby’ekihandiiko eki, reeba aha http://advances.sciencemag.org/cgi/ebirimu/omu bwijwire/7/3/eabe2631/DC1.
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Omuringo gw‘okushoboorora kw‘ahaiguru gw‘omutego gw‘omushana 1 ogugumire omu mwanya gw‘okugarukamu niguha okumanya okusya aha nkora ya quinone.
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