Aderesi ihari ubu: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Ikigo cy’Ubushakashatsi ku Binyabuzima mu by’Ubwenge.
Itsinda ry’urumuri rutunganya urumuri 1 (RC-LH1) ni ryo shingiro ry’ishusho rya bagiteri zikora ifototrophic y’umuhengeri. Twashyizeho imiterere ibiri ya mikorosikope ya cryo-electron ya RC-LH1 complex ikomoka kuri Rhodopseudomonas palustris. Imiterere ya 2.65-Å y’itsinda rya RC-LH114-W igizwe n’imirongo 14 ya LH1 ikikije RC, ihagarikwa na poroteyine W, mu gihe idafite poroteyine-W ari imiterere ya RC ikikije RC. Itsinda ry’imirongo 16 ya LH1 ifunze. Kugereranya iyi miterere bitanga ubumenyi ku mikorere ya quinone muri RC-LH1 complex, harimo n’impinduka zitari zaragaragajwe mbere iyo ihambiriye quinone ahantu ha RC QB, ndetse n’aho quinone ihambiriye, bifasha kuyishyira kuri RC. Imiterere yihariye ya poroteyine ya W ibuza ifunga rya LH1 log, bityo hagashyirwaho umuyoboro wo kwihutisha ihererekanya rya quinone/quinolone.
Ingufu zitangwa na fotositezi zishobora kubungabunga ubuzima hafi ya bwose ku isi, kandi zifite ubushobozi bukomeye bwo gukoresha ikoranabuhanga ry'izuba. Nubwo ziteza imbere fotositezi ku isi, bagiteri z'umuhengeri zigaragaza uburyo butandukanye bw'ingufu n'ubushobozi bwo guhindura imikorere y'umubiri. Zishobora kwirinda fotositezi no gukura kuko bagiteri z'umuzunguruko mu mwijima, zishobora gushyira azote na dioxyde de carbone, zigakora hydrogen, kandi zikangiza ibintu bihumura (1-3). Kugira ngo urumuri rutange ingufu muri ibi bikorwa, rugomba guhinduka vuba kandi neza ingufu za shimi. Iyi nzira itangira igihe antenna ifata urumuri yinjiza urumuri hanyuma ikayohereza ingufu zafashwe mu kigo cy'imyitozo (RC), bityo igatangira gutandukanya amashanyarazi (4 - 7). Igice cy'ibanze cya fotositezi ... Antene yoroshye ya LH1 igizwe na heterodimer 16 cyangwa 17 αβ zikikije RC (9-13) mu muyoboro ufunze, ariko mu zindi complexes z'ibanze, peptides za transmembrane zihagarika gukomeza kwa LH1 ikikije, bityo bigateza imbere ikwirakwira rya quinol/quinone hagati ya RC na cytochrome bc1 complex (11, 13-15). Ikimera cy'umuhengeri gifotora Rhodopseudomonas (Rps.) ni ikiremwa cy'icyitegererezo gishobora gusobanukirwa ingufu n'ihererekanya rya electron rishyigikira fotosite. Imiterere ya mbere ya kristu ya Rps. Icyitegererezo cya palustris RC-LH1 complex ni RC, ikikijwe n'imiyoboro 15 ya heterodimeric LH1, ihagarikwa na poroteyine itazwi yitwa "Poroteyine W" (14). Poroteyine-W yaje kumenyekana ko ari RPA4402, ikaba ari poroteyine idasanzwe ya 10.5kDa ifite helices eshatu za transmembrane (TMH) (16). Turasaba guhindura izina rya rpa4402 gene ihindura poroteyine W kuri pufW kugira ngo bihuze n'amazina akoreshwa kuri gene ihindura RC-L, M (pufL, pufM) na LH1α, β (pufA, pufB). Igishimishije ni uko poroteyine-W iboneka gusa muri 10% bya RC-LH1, bigaragaza ko Rps. palustris ikora complexes ebyiri zitandukanye za RC-LH1. Aha, turagaragaza imiterere ya cryo-EM (cryo-EM) ifite ubushobozi bwo hejuru bw'imiterere ibiri y'ingenzi, imwe ifite poroteyine W na heterodimers 14 za αβ, indi idafite poroteyine W na loop ya 16 ya Heterodimer LH1 ifunze. Imiterere yacu igaragaza impinduka mu gusobanukirwa complexe ya RC-LH1 ya Rps. palustris, kuko twasesenguye umubare w'abantu bahuje buri bwoko kandi dufite ubushobozi buhagije bwo gushyira neza buri peptide n'ibice bifatanye na lipide na quinones bifitanye isano. Igereranya ry'iyi miterere ryerekana ko poroteyine eshatu za TMH-W zitaraboneka mu yindi RC-LH1 complex kugeza ubu zikora umuyoboro wa quinone kugira ngo zihutishe ihererekanya rya quinone/quinolone. Ahantu hanini habikwa lipide na quinone hometse, kandi twagaragaje impinduka nshya nyuma yo guhuza quinone na RC, bishobora kuba bikwiriye kuri RC ya photosystem II (PSII) y'ibinyabuzima bifite ogisijeni. Ibyo twabonye bitanga ubumenyi bushya ku miterere y'ihererekanya rya quinone/quinolone n'ihererekanya mu ihuriro ry'ibanze rya RC-LH1 rya bagiteri ya purple phototrophic.
Kugira ngo tworohereze inyigo irambuye ku buryo bubiri buboneka muri Rps. palustris, dutandukanya buri RC-LH1 dukoresheje uburyo bwa biochemical. Poroteyine W-deficient complex (izwi hano nka ΔpufW) yasukuwe ku bwoko budafite gene ya pufW (16), kandi complex imwe gusa ya RC-LH1 niyo ishobora gukorwa. Poroteyine W-complex ikorwa n'ubwoko. Poroteyine W y'ubu bwoko ihindurwa na tag ya His inshuro 10 kuri C-terminus yayo, kugira ngo complex irimo poroteyine W ishobore guhuzwa neza na poroteyine W nyinshi zidafite binyuze mu guhagarika icyuma. Complex itandukanyijwe neza (16) Affinity Chromatography (IMAC).
Nkuko bigaragara ku Ishusho ya 1, complexes zombi zirimo sub-unit eshatu RC (RC-L, RC-M na RC-H) zikikijwe na antene ya LH1. Imiterere ya 2.80-A y'complex idafite protein-W igaragaza heterodimer 16 αβ, zikora umuyoboro wa LH1 ufunze ukikije RC, hano hiswe RC-LH116 complex. Imiterere ya 2.65Å y'complex irimo protein-W ifite 14-heterodimer LH1 ivangwa na protein-W, hano hiswe RC-LH114-W.
(A na B) Ishusho y'ubuso bw'ikinyabutabire. (C na D) Amabara afatanye agaragara mu nkoni. (E na F) Ibinyabutabire bibonwa mu buso bwa cytoplasmike bifite peptides na LH1 subunits bigaragazwa mu mashusho, kandi bihabwa inomero z'isaha uhereye ku cyuho cya poroteyine-W [bihuye n'imibare ya Rba. sphaeroides complex (13)]. Kuri LH1-α, ibara ry'igice cya poroteyine ni umuhondo; kuri LH1-β, ibara ry'igice cya poroteyine ni ubururu; kuri poroteyine-W, poroteyine ni umutuku; kuri RC-H, ni cyan; kuri RC-L, ni Orange; kuri RC-M, ni magenta. Ibinyabutabire bigaragazwa n'inkoni, icyatsi kibisi kigereranya BChl na BPh a molecules, umuhengeri ugereranya carotenoids, naho umuhondo uhagarariye molecules za UQ10. (G na H) Ishusho y'icyuho cya poroteyine-W mu gace kangana na RC-LH114-W complex (G) na RC-LH116 complex (H). Ibice bihuza imiterere y’umubiri bigaragazwa mu buryo bwo kuzuza umwanya, quinone ya chelated igaragazwa mu ibara ry’ubururu. Icyuho cya poroteyine-W kigaragazwa n’umurongo w’ubururu ucagaguye muri (G), naho utwobo duto aho quinone/quinolol ikwirakwira ku mpeta ya LH116 tugaragazwa n’umurongo w’umukara ucagaguye muri (H).
Ishusho ya 1 (A na B) igaragaza RC ikikijwe n'uduce dufunguye cyangwa dufunze twa heterodimer za LH1αβ, buri imwe ihuza BChl ebyiri na carotenoid imwe (Ishusho ya 1, C na D). Ubushakashatsi bwabanje bwagaragaje ko Rps ari complex ya LH1. Mu nzira ya biosynthetic ya spirulina xanthin, ubwo bwoko bugizwe n'ubwoko buvanze bwa carotenoids (17). Ariko, spiropyrroxanthin ni carotenoid ikomeye kandi ubucucike bwayo burashimishije. Kubwibyo, twahisemo gukora model ya spiroxanthin ahantu hose LH1 ihurira. Polypeptides za alpha na beta ni TMH imwe ifite uturere duto tw'inyuma twa membrane (Ishusho ya 1, A, B, E, na F). Nubwo ubucucike bw'ibisigazwa 17 kuri C-terminus butagaragaye, alpha polypeptide yaciwe kuva kuri Met1 kugera kuri Ala46 muri complexes zombi. β polypeptide yagabanutse kuva kuri Gly4 kugera kuri Tyr52 muri RC-LH116, no kuva kuri Ser5 kugera kuri Tyr52 muri RC-LH114-W. Nta bucucike bwa 3 cyangwa 4 N-terminal cyangwa 13 C-terminal byagaragaye (Ishusho S1). Isesengura rya Mass spectrometry rya complex ya RC-LH1 ivanze yakozwe mu bwoko bwa wild-type ryagaragaje ko agace kabuze kari ingaruka zo gucikamo kw'izi peptides (Ishusho S1 na S2). Ishusho ya N-terminal ya α-Met1 nayo yagaragaye (f). Isesengura ryagaragaje ko α-peptide igizwe n'ibisigazwa fMet1 kugera kuri Asp42/Ala46/Ala47/Ala50, naho β-peptide igizwe n'ibisigazwa Ser2 kugeza kuri Ala53, bihuye neza n'ikarita y'ubucucike bwa EM buri hasi.
Guhuza α-His29 na β-His36 bituma BChls irebana; buri heterodimer ya αβ iterana n'abaturanyi bayo kugira ngo ikore umuyoboro ufunguye (RC-LH114-W) cyangwa umuyoboro ufunze (RC-LH116) ukikije RC. Urutonde rw'amabara ahujwe na exciton (Ishusho ya 1, C na D). Ugereranije n'umurongo wa 877 nm wa RC-LH114-W, ihinduka ry'umutuku rya 880 nm rya RC-LH116 ni 3 nm (Ishusho ya 2A). Ariko, spectrum y'uruziga rwa dichroism ni hafi kimwe (Ishusho ya 2B), bigaragaza ko nubwo hari itandukaniro rigaragara hagati y'imiyoboro ifunguye n'ifunze, ibidukikije bya BChls bisa cyane. Ihinduka ry'umutuku rya absorption rishobora guterwa no kugabanuka k'ubushyuhe no kwiyongera kw'ubudahangarwa ku muyoboro ufunze (18, 19), impinduka mu guhuza ibara ryatewe n'umuyoboro ufunze (20, 21), cyangwa uruvange rw'izi ngaruka ebyiri (11).
(A) Interuro y’ubushyuhe bwa Ultraviolet/igaragara/igera hafi ya infrared, aho impera zayo zigaragazwa n’amabara ajyanye nayo kandi zigashyirwa ku rwego rwa BPh kuri 775 nm. (B) Interuro y’ubushyuhe bw’uruziga yashyizwe ku rwego rwa BChl kuri 805 nm. (C na D) Interuro ya ΔA yatoranijwe muri spectra y’ubushyuhe ya RC-LH114-W complex (C) na RC-LH116 complex (D). Kugira ngo bigereranywe neza, spectra zose zishyirwa ku rwego rwa ∆A ya −A kuri 0.2 ps. (E) Igipimo cya oxidation ya cytochrome c2 nyuma yo gushya mu gihe hari ibipimo bitandukanye bya UQ2 (reba Ishusho S8 ku makuru fatizo). (F) Mu turemangingo twakuriye munsi y’urumuri ruto, ruri hagati cyangwa ruri hejuru (10, 30 cyangwa 300μMm-2 s-1, uko bikurikirana), poroteyine W na RC-L bigizwe mu rwego rwa complex isukuye n’igipimo cya membrane cyatandukanyijwe. Menya urwego rwa poroteyine ukoresheje electrophoresis ya SDS-polyacrylamide gel na immunophoresis (reba Ishusho S9 kugira ngo ubone amakuru y’ibanze). Menya ikigereranyo ugereranyije n’icunga rya RC-LH114-W ryasukuwe. Igipimo cya stoichiometric cya RC-L na poroteyine-W y’icunga ni 1:1.
BChls ziri ku mwanya wa 1 mu ruziga rwa αβ14 rwa RC-LH114-W (Ishusho ya 1, A, C, na E) zegereye umutanga-nguzo w’ibanze wa RC (P) kuri 6.8Å kurusha BChls zingana muri RC-LH116 (Ishusho ya 1, B, D, na F, na Ishusho ya S3); icyakora, kinetics y’ingufu z’igihe gito z’ibice bibiri bigaragaza ko kuri RC-LH114-W na RC-LH116, ibipimo by’igihe cyo guhererekanya ingufu kuva kuri LH1 kugera kuri RC ari 40 ±4 na 44±3 ps (Ishusho ya 2). , C na D, Ishusho ya S4 na Imbonerahamwe ya S2). Nta tandukaniro rigaragara mu guhererekanya ingufu muri RC (Ishusho ya S5 n’inyandiko y’inyongera ijyanye nayo). Turakeka ko imikoranire ya hafi y’igihe cyo guhererekanya ingufu hagati ya LH1 na RC-P biterwa n’intera, inguni n’ingufu zishobora kuba zingana za BChl nyinshi muri ruziga ebyiri za LH1. Bigaragara ko gusuzuma imiterere y'ingufu za LH1 kugira ngo ugere ku ntera ntoya bitakwihuta kurusha kwimura ingufu zitaziguye uvuye ahantu hato ujya kuri RC. Uruziga rwa LH1 rworoshye muri RC-LH114-W rushobora kandi kunyura mu bushyuhe buke mu gihe cy'ubushyuhe buke kugira ngo rusesengurwe, kandi hari imiterere miremire ya αβ14 ku bushyuhe bw'icyumba uvuye ku ntera ya βBChls ku mwanya wa RC 1.
Urusobe rwa RC-LH116 rugizwe na BChls 32 na carotenoids 16, kandi imiterere yarwo muri rusange ni imwe n’iyavuye muri Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 strain (PDB ID 7C9R) (12) na algae y’icyatsi kibisi (Blc.viridis) (PDB ID 6ET5) (10). Nyuma yo guhuza, hagaragaye gusa itandukaniro rito mu myanya ya heterodimer za αβ, cyane cyane 1-5, 15, na 16 (Ishusho S6). Kuba hariho poroteyine-W bigira ingaruka zikomeye ku miterere ya LH1. TMHs zayo eshatu zihujwe n’imirongo migufi, aho N-terminal iri ku ruhande rwa lumen rw’urusobe na C-terminal iri ku ruhande rwa cytoplasmic (Ishusho 1A na 3, A kugeza D). Poroteyine-W ahanini irangwa no kudahumeka (Ishusho ya 3B), kandi TMH2 na TMH3 bikorana na LH1αβ-14 kugira ngo bikore ubuso bwa transmembrane (Ishusho ya 3, B na E kugeza kuri G). Ahantu hahurira ahanini hagizwe n'ibisigazwa bya Phe, Leu na Val mu gace ka transmembrane. Ibi bisigazwa bikusanyirijwe hamwe na aside amine ziterwa no kudahumeka na αβ-14. Hari ibisigazwa bimwe na bimwe byo mu gice cy'inyuma nabyo bigira uruhare mu mikoranire, harimo n'umugozi wa hydrogen hagati ya W-Thr68 na β-Trp42 ku buso bw'aho hantu hahurira (Ishusho ya 3, F na G). Ku buso bwa cytoplasm, Gln34 iri hafi y'itsinda rya keto rya carotenoids za αβ-14. Byongeye kandi, molekile ya n-dodecyl β-d-maltoside (β-DDM) yarakemutse, maze umurizo wayo uterwa no kudahumeka waragutse ukagera ku ihuriro riri hagati ya poroteyine-W na αβ-14, kandi umurizo wa lipide ushobora kuba uri mu mubiri. Twabonye kandi ko uturere twa C-terminal twa poroteyine W na RCH turi hafi cyane, ariko ntabwo turi mu rwego rwo gukora imikoranire yihariye (Ishusho ya 1, A na E). Ariko, hashobora kubaho imikoranire muri aside amine C-terminal z'izi poroteyine ebyiri zitarakemuka, zishobora gutanga uburyo bwo kwinjizwa kwa poroteyine-W mu gihe cyo guteranya RC-LH114-W.
(A) Poroteyine-W, ireba aho LH1αβ14 ihurira mu ishusho ya katuni, ifite uruhande rumeze nk'uruti (umutuku), rugaragara mu gice cy'igishushanyo cy'amashanyarazi (ubuso bw'imvi bugaragara neza bufite urwego rwa 0.13). (B) Poroteyine-W igaragazwa n'ubuso bw'ibara rya hydrophobic. Uduce tw'izuba n'utw'amashanyarazi tugaragara mu buryo bwa cyan, uduce twa hydrophobic tugaragara mu buryo bw'umweru, naho uduce twa hydrophobic cyane tugaragara mu buryo bwa orange. (C na D) Poroteyine-W igaragazwa mu buryo bwa katuni, icyerekezo cyayo ni kimwe n'icya (A) (C), kandi izunguruka na 180° (D). Dukurikije aho iri muri uru rutonde, ibisigazwa bishobora kumenyekana bifata ibara ry'umukororombya, aho N-terminal ari ubururu na C-terminal ari umutuku. (E) Poroteyine-W mu buryo bumwe n'ubwa (A), kandi ibisigazwa biri ku buryo bwa protein-W:LH1 bigaragazwa n'udukoni dufite ibimenyetso bifatanye. (F) Poroteyine-W izunguruka 90° ugereranije na (E) na LH1αβ14 mu ishusho y'ibishushanyo, kandi ugereranije n'ibisigazwa by'aho bihurira mu ishusho y'umurongo. Ibisigazwa birebire biva kuri polipeptide ya beta byanditseho. Cofactor igaragazwa nk'umurongo uhuye n'ibara ry'Ishusho ya 1, β-DDM yangiritse igaragazwa mu ibara ry'umukara, naho ogisijeni igaragazwa mu ibara ry'umutuku. (G) Ishusho muri (F) izunguruka 180°, hamwe n'ibisigazwa bigaragara bya polipeptide ya alpha.
Poroteyine-W isimbuza heterodimer ya αβ (iya 15 ku Ishusho ya 1F), bityo ikarinda ko umugozi ufunga no kuzunguruka heterodimer eshatu za mbere za αβ. Byagaragaye ko inguni ntarengwa yo kuzunguruka ya heterodimer ya mbere ya αβ-1 ugereranije n'ishusho isanzwe yari 25° kugeza 29° (Ishusho ya 1, A na E), yakozwe n'inguni ya αβ-1 ya 2° kugeza 8° muri RC A itandukanyije cyane - LH116 (Ishusho ya 1, B na F). Heterodimer ya kabiri n'iya gatatu zizunguruka kuri 12° kugeza 22° na 5° kugeza 10°, uko bikurikirana. Kubera imbogamizi ya RC, kuzunguruka kwa αβ-1 ntabwo kurimo kabiri ka αβ (gahuye na αβ ya 16 ku Ishusho ya 1F), bityo hakagaragara icyuho mu ruziga rwa LH1 (Ishusho ya 1, A na E). Bitewe n'ibura rya heterodimer ebyiri za αβ, ziherekejwe no gutakaza BChl enye na carotenoide ebyiri, nta na kimwe muri carotenoide gifatana n'igice cya αβ-1 cyazungurutse, bigatuma habaho impeta ya LH114-W irimo carotenoide 13 za Vegetarian na BChls 28. Isuzuma ry'imiterere y'aho hantu ry'ibice bibiri biri mu turere twa αβ1 kugeza kuri 7 riri hasi ugereranije n'iry'ibindi bice bya LH1, bishobora kugaragaza imiterere y'igice cya LH1 kiri hafi y'aho RC QB iherereye (Ishusho ya 4).
Amafoto ya RC-LH114-W (A na B) na RC-LH116 (C na D) agaragara mu buryo bumwe bwo hejuru/uruhande (A na B) (A na C) n'ubuso bw'umwobo bw'Ishusho ya 1. (B na D). Imfunguzo z'amabara zigaragara iburyo.
Indi ngingo y’ingenzi iranga imiterere ifite igipimo cya stoichiometric cya 1:14 ni Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Ariko, poroteyine W na PufX nta homology igaragara bifite, kandi bigira ingaruka zikomeye ku miterere ya LH1. PufX ni TMH imwe ifite domaine ya N-terminal cytoplasmic ikorana n'uruhande rwa cytoplasmic rwa RC-H subunit (13) ku mwanya uhuye na Rps. palustris LH116αβ-16. PufX ikora umuyoboro w'ihererekanya rya quinone/quinolone hagati ya RC-LH1 na cytochrome bcl complex kandi iboneka muri Rba. sphaeroides core complex yose (13). Nubwo ihuriro rya monomer-monomer riri muri Rba. Dimer ya sphaeroides RC-LH1-PufX iherereye aho poroteyine W ihurira muri RC-LH114-W, kandi icyuho giterwa na PufX na poroteyine-W kiri ahantu hangana (Ishusho S7A). Icyuho kiri muri RC-LH114-W gihuzwa kandi n'umuyoboro wa quinone (8) wa Pseudomonas rosea LH1, ukorwa na peptides zidafitanye isano na poroteyine W cyangwa PufX (Ishusho S7B). Byongeye kandi, umuyoboro wa quinone muri Blc. LH1 y'icyatsi kibisi ya emerald yakozwe no gukuramo igice kimwe cya γ (7) iri ahantu hangana (Ishusho S7C). Nubwo ihuzwa na poroteyine zitandukanye, kugaragara kw'iyi miyoboro ya quinone/quinolol mu mwanya uhuriweho muri RC-LH1 complex bisa nkaho ari urugero rw'ihindagurika ry'ibihe, bigaragaza ko icyuho giterwa na poroteyine W gishobora gukora nk'umuyoboro wa quinone.
Icyuho kiri mu ruziga rwa LH114-W cyemerera ikorwa ry’agace k’urukiramende gahoraho hagati y’umwanya w’imbere wa RC-LH114-W complex n’urukiramende rw’umubyimba (Ishusho ya 1G), aho guhuza uturere tubiri binyuze mu mwobo wa poroteyine nk’uko bimeze muri poroteyine. Urukiramende rwa RC-LH116 rusa n’urukiramende rufunze rwa Tch. (22) (Ishusho ya 1H). Kubera ko gukwirakwira kwa quinone binyuze mu ruziga kwihuta kuruta gukwirakwira binyuze mu muyoboro muto wa poroteyine, uruziga rwa LH114-W rufunguye rushobora gutuma RC ihinduka vuba kuruta uruziga rwa LH116 rufunze, kandi gukwirakwira kwa quinone muri RC bishobora kuba bito. Kugira ngo tumenye niba poroteyine W igira ingaruka ku ihinduka rya quinones binyuze muri RC, twakoze isuzuma rya cytochrome oxidation ku gipimo runaka cya ubiquinone 2 (UQ2) (ikigereranyo cya UQ10 karemano gifite umurizo mugufi wa isoprene) (Ishusho ya 2E). Nubwo kuba hariho quinone ya chelated bibangamira kumenya neza ko Michaelis igaragara (RC-LH114-W na RC-LH116 bikwiranye na 0.2±0.1μM na 0.5±0.2μM, uko bikurikirana), igipimo ntarengwa cya RC-LH114-W (4.6±0.2 e-RC-1 s-1) ni 28±5% kuruta RC-LH116 (3.6±0.1 e-RC-1 s-1).
Twabanje kugereranya ko poroteyine-W iboneka hafi 10% by'insinga z'umubiri (16); hano, igipimo cy'ubucucike bw'uturemangingo tw'urumuri ruto, urumuri ruciriritse, n'urumuri rwinshi ni 15±0.6%, 11±1% na 0.9±0.5, uko bikurikirana % (Ishusho ya 2F). Igereranya ry'ingano rya spectrometry y'ubucucike ryagaragaje ko kongeramo histidine tag bitagabanyije ubwinshi bwa poroteyine-W ugereranije n'ubwoko bw'ubwoko bw'ishyamba (P = 0.59), bityo izi ntera si ikintu cyahinduwe cya poroteyine-W (Ishusho ya S10). Ariko, ubu bucucike buke bwa poroteyine-W muri RC-LH1 complex bushobora gutuma zimwe muri RC zihinduka ku muvuduko wihuta, bityo bikagabanya ihindagurika rya quinone/quinolone rigenda buhoro muri RC-LH116 complex. Twabonye ko igipimo cy'ubucucike bw'urumuri rwinshi kidahuye n'amakuru aherutse gutangwa muri transcriptomics, agaragaza ko igaragara rya gene ya pufW ryiyongera iyo urumuri rukomeye (Ishusho ya S11) (23). Itandukaniro riri hagati ya pufW transcription na protein-W ishyirwa muri RC-LH1 complex ni urujijo kandi rishobora kugaragaza uburyo poroteyine igenzurwamo cyane.
Muri RC-LH114-W, 6 cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) na 29 β-DDM molekile zashyizwemo kandi zigashyirwamo icyerekezo cyazo 6 CDL, 24 POPC, 2 POPG na 12 βDDM. RC-LH116 (Ishusho ya 5, A na B). Muri izi ngingo ebyiri, CDL iri hafi ku ruhande rwa cytoplasmic rw'urusobe rw'imitsi, mu gihe POPC, POPG na β-DDM ahanini ziherereye ku ruhande rw'urusobe rw'imitsi. Molekile ebyiri za lipide na detergent zakuwe mu gace ka αβ-1 kugeza kuri αβ-6 k'urusobe rwa RC-LH114-W (Ishusho ya 5A), naho eshanu zakuwe mu gace kangana na RC-LH116 (Ishusho ya 5B). Hari izindi lipide nyinshi zabonetse ku rundi ruhande rw'urusobe rw'imitsi, cyane cyane CDL, zakusanyirijwe hagati ya RC na αβ-7 na αβ-13 (Ishusho ya 5, A na B). Izindi lipide na sabuni zakuwemo mu miterere ziherereye hanze ya LH1 ring, kandi iminyururu ya acyl yakuwemo neza iri hagati ya LH1 subunits, byemewe ko ari β-DDM muri RC-LH114-W, kandi isobanurwa ko ari β-DDM muri RC A uruvange rwa β-DDM na POPC-LH116. Imiterere isa ya lipide na sabuni za chelating mu miterere yacu igaragaza ko ari ahantu hafite akamaro mu mikorere (Ishusho ya S12A). Imiterere ya molecule zingana muri Tch nayo ifite imiterere myiza. Yoroshye kandi irasobanutse. Ubwoko bwa 970 RC-LH1s (Ishusho S12, B kugeza E) (9, 12) hamwe n'ibisigazwa bya hydrogen-bonding byo mu itsinda ry'umutwe wa lipide byagaragaje ko bibungabunga neza mu buryo bwo guhuza urukurikirane (Ishusho S13), bigaragaza ko Conserved CDL ifatanye na RC (24), izi mbuga zishobora kubungabungwa muri RC-LH1 complex.
(A na B) Peptide za RC-LH114-W (A) na RC-LH116 (B) zigaragazwa n'udushushanyo, kandi ibara ryazo rigaragazwa n'udukoni, hakoreshejwe ishusho y'amabara iri ku Ishusho ya 1. Lipide zigaragazwa mu ibara ritukura, naho isabune zigaragazwa mu ibara ry'umukara. UQ ihujwe n'ahantu ha RC QA na QB ni umuhondo, mu gihe UQ yihariye ari ubururu. (C na D) Ishusho imwe na (A) na (B), hamwe na lipide zitarimo. (E kugeza kuri G) Ishusho yagutse ya Q1 (E), Q2 (F) na Q3 (G) yavuye kuri RC-LH116, hamwe n'iminyururu yo ku ruhande igira ingaruka ku yindi. Iminyururu ya hydrogen igaragazwa nk'imirongo y'umukara iciyemo uduce.
Muri RC-LH116, RC QA na QB UQ byombi, bigira uruhare mu kohereza electron mu gikorwa cyo gutandukanya charge, birangirika ahantu bihurira. Ariko, muri RC-LH114-W, QB quinone ntabwo yarakemutse kandi bizaganirwaho birambuye hepfo. Uretse QA na QB quinones, molekile ebyiri za UQ zifite chelate (ziri hagati ya RC na LH1 rings) zishyirwa mu miterere ya RC-LH114-W hakurikijwe amatsinda yazo y’umutwe yakemutse neza (aherereye muri Q1 na Q2, uko bikurikirana). Ishusho ya 5C). Ibice bibiri bya isoprene byashyizwe kuri Q1, kandi ikarita y’ubucucike ikemura imirizo 10 yose ya isoprene ya Q2. Mu miterere ya RC-LH16, molekile eshatu za UQ10 zifite chelate (Q1 kugeza Q3, Ishusho ya 5D) zarakemutse, kandi molekile zose zifite ubucucike busobanutse mu murizo wose (Ishusho ya 5, D kugeza G). Muri izo nyubako zombi, aho imitwe ya quinone ya Q1 na Q2 iherereye bifite imiterere myiza (Ishusho S12F), kandi bikorana na RC gusa. Q1 iherereye ku muryango w'icyuho cya W cya RC-LH114-W (Ishusho 1G na 5, C, D na E), naho Q2 iherereye hafi y'aho QB ihurira (Ishusho 5, C, D) na F). Ibisigazwa bya L-Trp143 na L-Trp269 byabitswe biri hafi cyane ya Q1 na Q2 kandi bitanga imikoranire ishobora gutandukana (Ishusho 5, E na F, na Ishusho S12). L-Gln88, 3.0 Å uvuye kuri ogisijeni ya kure ya Q1, itanga umugozi ukomeye wa hydrogen (Ishusho 5E); ibi bisigazwa bibitswe muri RC zose uretse isano iri kure cyane (Ishusho S13). L-Ser91 yasimbuwe mu buryo burambye na Thr mu zindi RC nyinshi (Ishusho S13), ni 3.8 Angstroms zikomoka kuri ogisijeni ya methyl ya Q1, kandi zishobora gutanga imigozi idakomeye ya hydrogen (Ishusho ya 5E). Q3 isa nkaho nta mikoranire yihariye ifite, ariko iherereye mu gace ka hydrophobic hagati ya RC-M subunit na LH1-α subunit 5 kugeza kuri 6 (Ishusho ya 5, D na G). Q1, Q2 na Q3 cyangwa quinones zegeranye nazo zarakemutse muri Tch. Gentle, Trv. Strain 970 na Blc. Imiterere ya iris (9, 10, 12) yerekana ahantu habikwa quinone y'inyongera muri RC-LH1 complex (Ishusho ya S12G). UQ eshanu zangiritse muri RC-LH116 zihuye neza na 5.8±0.7 ya buri complex igenwa na high performance liquid chromatography (HPLC), mu gihe UQ eshatu zangiritse muri RC-LH114-W ziri hasi ugereranyije n'agaciro kapimwe ka 6.2±0.3 (Ishusho S14) kerekana ko hari molekile za UQ zidakemutse mu miterere.
Polypeptides za L na M zigizwe na TMH eshanu kandi zikora heterodimer ihuza dimer imwe ya BChl, monomers ebyiri za BChl, monomers ebyiri za bacteriophage (BPh), na molekile imwe itari Heme na molekile imwe cyangwa ebyiri za UQ10. Binyuze mu kuba hari imigozi ya hydrogen ku itsinda rya ketone rigizwe n'ibice byayo bizwi kandi bizwi muri Rps, carotenoids zishyirwa muri M-subunit, yitwa cis-3,4-dehydroorhodopin. Ubwoko (25). Igice cy'inyuma cya RC-H gifatanye na membrane na TMH imwe. Imiterere ya RC muri rusange isa na subunit eshatu za RC z'ubwoko bufitanye isano (nka Rba) sphaeroides (PDB ID: 3I4D). Macrocycles za BChl na BPh, umugongo wa carotenoid n'icyuma kitari heme bihurirana mu rwego rwo gusubiza ibi binyabuzima, kimwe n'itsinda rya UQ10 ku rubuga rwa QA na QB quinone kuri RC-LH116 (Ishusho S15).
Kuba hariho imiterere ibiri ya RC ifite igipimo gitandukanye cy’ubwiyongere bw’aho QB iherereye bitanga amahirwe mashya yo gusuzuma impinduka zihoraho zijyana no gufatanya kwa QB quinone. Muri RC-LH116 complex, QB quinone iherereye mu mwanya wa "proximal" (26), ariko gutandukana kwa RC-LH114-W nta quinone ya QB ifite. Nta quinone ya QB iboneka muri RC-LH114-W, ibi biratangaje kuko complex ikora, kurusha RC-LH116 complex ifite quinone ya QB ifite imiterere. Nubwo impeta ebyiri za LH1 zigera kuri quinone esheshatu, eshanu zirakemuka mu miterere ya RC-LH116 ifunze, mu gihe eshatu gusa ari zo zifite imiterere mike mu miterere ya RC-LH114-W ifunguye. Uku kwiyongera kw’imiterere y’imiterere gushobora kugaragaza gusimburwa vuba kw’ahantu ha RC-LH114-W QB, kinetics ya quinone yihuta mu miterere, ndetse n’amahirwe menshi yo kwambuka umuyoboro wa LH1. Turasaba ko kubura kwa UQ mu gice cya RC QB cya RC-LH114-W bishobora kuba biterwa n’uburyo bugoye kandi bukora cyane, kandi ahantu ha QB ha RC-LH114-W hahise hahagarara mu ihindagurika rya UQ. Icyiciro cyihariye (aho kwinjira mu gice cya QB cyafunzwe) kigaragaza imiterere y’iki gikorwa.
Nta QB, kuzenguruka kwa L-Phe217 ku mwanya udahuye na UQ10, kuko bizatera impanuka y'ahantu hamwe n'igice cya mbere cya isoprene cy'umurizo (Ishusho ya 6A). Byongeye kandi, impinduka z'ingenzi zigaragara ziragaragara, cyane cyane helix de (helix ngufi iri hagati ya TMH D na E) aho L-Phe217 yimurirwa mu mufuka w'umuhuza wa QB n'izunguruka rya L-Tyr223 (Ishusho ya 6A) Kugira ngo hameneke umugozi wa hydrogen hamwe na framework ya M-Asp45 kandi hagafungwa umuryango w'aho QB yimurirwa (Ishusho ya 6B). Helix de izunguruka ku gice cyayo, Cα ya L-Ser209 yimuwe na 0.33Å, mu gihe L-Val221Cα yimuwe na 3.52Å. Nta mpinduka zigaragara muri TMH D na E, zishobora kuba zirenzeho mu miterere yombi (Ishusho ya 6A). Uko tubizi, iyi ni yo miterere ya mbere muri RC karemano ifunga aho QB iherereye. Kugereranya n'imiterere yuzuye (iboheshejwe na QB) bigaragaza ko mbere yuko quinone igabanuka, hakenewe impinduka mu miterere yayo kugira ngo yinjire muri quinone. L-Phe217 irazenguruka igakora imikoranire ya π-stacking n'itsinda ry'umutwe wa quinone, maze helix ijya hanze, bigatuma igishishwa cya L-Gly222 n'urunigi rwo ku ruhande rwa L-Tyr223 birema umuyoboro wa hydrogen bond ufite imiterere ihamye ya hydrogen bond (Ishusho ya 6, A na C).
(A) Ishusho ihuza ya hologram (umunyururu wa L, umunyururu wa orange/M, magenta) na apo (imvi), aho ibisigazwa by'ingenzi byerekanwa mu buryo bw'ishusho isa n'inkoni. UQ10 igaragazwa n'umuhondo. Umurongo w'utudomo ugaragaza imigozi ya hydrogen igizwe n'imiterere yose. (B na C) Ishusho y'ubuso bwa apolipoprotein n'imiterere yose y'impeta, igaragaza umwuka wa ogisijeni wo ku ruhande rwa L-Phe217 mu bururu na L-Tyr223 mu ibara ry'umutuku, uko bikurikirana. Igice cya L ni umuhondo; ibice bya M na H ntabwo bifite ibara. (D na E) Apolipoprotein (D) n'ahantu hose (E) RC QB [ibara hakurikijwe (A) uko bikurikirana] na Thermophilus thermophilus PSII (icyatsi kibisi, ubururu hamwe na quinone ya pulasitiki; PDB ID: 3WU2) Kunganya (58).
Mu buryo butunguranye, nubwo hari imiterere myinshi ya RC zidafite QB zidafite LH1, impinduka zagaragaye muri ubu bushakashatsi ntizatangajwe mbere. Izi zirimo imiterere ya QB iva kuri Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) na Rba. sphaeroides (PDB ID: 1OGV) (29), zose zikaba zisa hafi n'imiterere yazo muri rusange ya QB. Isuzuma ryimbitse rya 3PRC ryagaragaje ko molekile za LDAO (Lauryl Dimethyl Amine Oxide) zifata ku muryango w'aho QB iherereye, bishobora kubuza kongera guhindurwa mu buryo bufunze. Nubwo LDAO idahinduka ahantu hamwe muri 1EYS cyangwa 1OGV, izi RC zitegurwa hakoreshejwe isabune imwe bityo zishobora gutanga ingaruka zimwe. Imiterere ya kristu ya Rba. Sphaeroides RC yahujwe na cytochrome c2 (PDB ID: 1L9B) nayo isa nkaho ifite ahantu hafunganye ha QB. Ariko, muri iki gihe, agace ka N-terminal ka polipeptide ya RC-M (gakorana n'ahantu hafunganye ha QB binyuze mu mugozi wa H w'ibisigazwa bya Tyr kuri Q helix) gafata imiterere idasanzwe, kandi impinduka ya QB ntabwo ikomeje gucukumburwa (30). Igishimishije ni uko tutarabona ubwoko nk'ubu bwa polipeptide ya M mu miterere ya RC-LH114-W, hafi kimwe n'agace ka N-terminal ka RC-LH116 RC. Tugomba kandi kumenya ko nyuma yo gukuraho antene ya LH1 ishingiye ku isukari, apolipoprotein RCs muri PDB zarakemutse, bikuraho ibiyaga bya quinone imbere na lipide mu cyuho kiri hagati ya RC n'imbere mu mpande ya LH1 ikikije (31, 32). RC ikomeza gukora kuko igumana ibintu byose bihuza, usibye QB quinone ishobora kubora, idakomeye kandi ikunze kubura mu gihe cyo kuyitegura (33). Byongeye kandi, birazwi ko gukuraho LH1 na lipide karemano zikora kuri RC bishobora kugira ingaruka ku mikorere, nko kugabanya igihe cyo kubaho kwa P+QB-state itandukanyijwe na charge (31, 34, 35). Kubwibyo, dutekereza ko kuba hari impeta ya LH1 ikikije RC bishobora kugumana aho QB "ifungiye", bityo bikarinda ibidukikije byo hafi ya QB.
Nubwo apolipoprotein (idafite QB quinone) n'imiterere yuzuye bigaragaza amashusho abiri gusa y'ihindagurika ry'ahantu ha QB, aho kuba urukurikirane rw'ibintu, hari ibimenyetso byerekana ko ihambira rishobora gukingirwa kugira ngo hirindwe kongera kwihambira hakoreshejwe hydroquinone kugira ngo hirindwe ikumirwa ry'ubutaka. Imikoranire ya quinolol na quinone hafi y'aho QB ya apolipoprotein ishobora kuba itandukanye, bigatuma RC itayifata. Byavuzwe kuva kera ko impinduka mu miterere zigira uruhare mu gufatana no kugabanya quinones. Ubushobozi bwa RCs zakonje bwo kugabanya quinones nyuma yo kwijima kwangiritse (36); X-ray crystallography igaragaza ko uku kwangirika guterwa na quinones za QB zifatiwe mu miterere "ya kure" hafi 4.5 Å uvuye aho hafi (26), 37). Turatanga igitekerezo cy'uko iyi miterere ya kure ari ishusho y'imiterere iri hagati ya apolipoprotein n'imiterere y'impeta yuzuye, ikurikira imikoranire ya mbere na quinone no gufungura aho QB ifunguye.
Ubwoko bwa II RC buboneka muri PSII complex ya bagiteri zimwe na zimwe zikora kuri fototrophic na cyanobacteria, algae n'ibimera bufite uburyo bwo kubungabunga imiterere n'imikorere (38). Uburyo imiterere igaragara ku Ishusho ya 6 (D na E) bushimangira isano iri hagati ya PSII RCs n'ahantu hazwi nka QB h'aho bagiteri RC complex iherereye. Iri gereranya ryabaye icyitegererezo cyo kwiga uburyo bufitanye isano bwa quinone ifatana n'igabanuka. Inyandiko zabanje zagaragaje ko impinduka mu miterere zijyana no kugabanya quinones kuri PSII (39, 40). Kubwibyo, ukurikije uburyo RC yakomeje kubungabunga, ubu buryo bwo gufatana butari bwaragaragaye mbere bushobora no gukoreshwa ku hantu hazwi nka QB h'ibimera bikora kuri fototrophic bifite ogisijeni.
Rps ΔpufW (gusiba pufW bitanditseho) na PufW-His (C-terminal 10x His-tagged protein-W ituruka ku bwoko bwa pufW locus karemano). palustris CGA009 yasobanuwe mu kazi kacu ka mbere (16). Ubu bwoko n'ubwoko bwa isogenic wild-type parent byakuwe muri firigo binyuze mu gukurura uturemangingo duto kuri PYE (buri litiro 5 g -1) (bibitswe muri LB kuri -80 °C, birimo 50% (w/v) glycerol) poroteyine, ivange ry'umusemburo na succinate) agar [1.5% (w/v)]. Isahani yashyizwe mu mwijima ijoro ryose mu gihe cy'ubushyuhe bw'icyumba mu gihe cy'umwuka udakora, hanyuma imurikirwa n'urumuri rwera (~50 μmolm-2 s-1) rwatanzwe na OSRAM 116-W halogen bulbs (RS Components, UK) mu gihe cy'iminsi 3 kugeza kuri 5 kugeza igihe ikoloni imwe igaragaye. Itsinda rimwe ryakoreshejwe mu gutera mililitiro 10 za M22+ (41) ziyongereyeho aside casamino ya 0.1% (w/v) (izwi hano nka M22). Iyi nkomoko yahinzwe mu gihe cy'umwuka wa ogisijeni nke mu mwijima kuri dogere selisiyusi 34, ikanyeganyega kuri 180 rpm mu gihe cy'amasaha 48, hanyuma mililitiro 70 z'iyi nkomoko ziterwa mu gihe nk'icyo mu gihe cy'amasaha 24. Iyi nkomoko ifite ingano ya mililitiro 1 ikoreshwa mu gutera mililitiro 30 za M22 mu icupa ry'ikirahure rya mililitiro 30 rikoresha screw-top transparent hanyuma ikarangizwa n'imirasire (~50μmolm-2 s-1) mu gihe cy'amasaha 48 hakoreshejwe imbaraga za magnetic Stirring rod. Hanyuma mililitiro 30 z'iyi nkomoko zatewe litiro 1 ya nkomoko mu gihe nk'icyo, hanyuma ikoreshwa mu gutera litiro 9 za nkomoko zamuritswe kuri ~200 μmolm-2 s-1 mu gihe cy'amasaha 72. Uturemangingo twakusanyijwe hakoreshejwe centrifugation kuri 7132 RCF mu gihe cy'iminota 30, twongera gushyirwa mu cyuma kiri kuri ~10 ml ya 20 mM tris-HCl (pH 8.0), hanyuma tubikwa kuri -20°C kugeza igihe bikenewe.
Nyuma yo gushonga, ongeramo utubumbe twa deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) n'ibinini bibiri bya Roche holoenzyme protease inhibitor (Merck, UK) ku tubumbe twasubijwemo. Mu tubumbe twa psi 20.000 (Aminco, USA), utwo tubumbe twahungabanye inshuro 8 kugeza kuri 12. Nyuma yo gukuraho uturemangingo tutavunitse n'imyanda idashonga binyuze mu gupima kuri 18.500 RCF mu gihe cy'iminota 15 kuri 4°C, uturemangingo twakuwe muri lysate ifite ibara binyuze mu gupima kuri 113.000 RCF mu gihe cy'amasaha 2 kuri 43.000°C. Kuraho igice gishonga hanyuma wongere ushyireho uturemangingo tw'amabara muri mililitiro 100 kugeza kuri 200 za 20 mM tris-HCl (pH 8.0) hanyuma uhindure homogene kugeza igihe nta mpande zigaragara zihari. Ururenda rw'amazi rwashyizwe mu cyuma cya tris-HCl 20 mM (pH 8.0) (Anatrace, USA) kirimo 2% (w/v) β-DDM mu gihe cy'isaha 1 mu mwijima kuri 4°C ubyutse buhoro buhoro. Hanyuma ukoresheje centrifuge kuri 70°C kugira ngo ushongeshe 150.000 RCF kuri 4°C mu gihe cy'isaha 1 kugira ngo ukureho ibisigaye bitashongesha.
Uduce dushonga twavuye mu bwoko bwa ΔpufW twashyizwe ku nkingi ya 50 ml ya DEAE Sepharose ion exchange column ifite inkingi eshatu (CV) za binding buffer [20 mM tris-HCl (pH 8.0) irimo 0.03% (w / v) β-DDM]. Oza inkingi ukoresheje CV binding buffers ebyiri, hanyuma woze inkingi ukoresheje 50 mM NaCl. RC-LH116 complex yakuweho hakoreshejwe linear gradient ya 150 kugeza 300 mM NaCl (muri binding buffer) kuri 1.75 CV, naho indi bindi bindi complex yakuweho hakoreshejwe bindi bindi irimo 300 mM NaCl kuri 0.5 CV. Kusanya imiterere y’ingufu iri hagati ya 250 na 1000 nm, komeza igice gifite igipimo cyo kwinjizwamo (A880/A280) kirenze 1 kuri 880 kugeza 280 nm, ugishyire mu mazi kabiri mu gice cyo kwihambira, hanyuma ukoreshe uburyo bumwe kuri inkingi ya DEAE On purification. Shyira ibice hamwe n’igipimo cya A880/A280 kiri hejuru ya 1.7 na A880/A805 kiri hejuru ya 3.0, kora icyiciro cya gatatu cy’ihindagurika rya iyoni, kandi ugumane ibice bifite igipimo cya A880/A280 kiri hejuru ya 2.2 na A880/A805 kiri hejuru ya 5.0. Iyi complex yasukuwe igice yashyizwe kuri ~2 ml muri Amicon 100.000 molekile cut-off (MWCO) centrifugal filter (Merck, UK), hanyuma ishyirwa ku gice cya Superdex 200 16/600 (GE Healthcare, US) kirimo 200 mM NaCl buffer, hanyuma ishyirwa muri buffer imwe kuri 1.5 CV. Kura spectra y'ingufu z'ingano, hanyuma ushyire spectra y'ingufu z'ingufu hamwe na A880/A280 ratios hejuru ya 2.4 na A880/A805 ratios hejuru ya 5.8 na 100 A880, hanyuma uhite uyikoresha mu gutegura cyangwa kubika cryo-TEM grid. Bika kuri -80°C kugeza bibaye ngombwa.
Ururenda rushongesha rwavuye muri PufW-His rwashyizwe ku gipimo cya 20 ml cya HisPrep FF Ni-NTA Sepharose (20 mM tris-HCl (pH 8.0) kirimo 200 mM NaCl na 0.03% (w/w)) muri IMAC buffer (GE Healthcare). v) β-DDM]. Igipimo cyogejwe na CV eshanu za IMAC buffer, hanyuma hashyirwaho CV eshanu za IMAC buffer irimo 10 mM histidine. Igice cy'ingenzi cyakuwe ku gipimo hamwe na IMAC buffer eshanu zifite 100 mM histidine. Igice kirimo RC-LH114-W complex gishyirwa kuri ~10 ml mu gikoresho cyavanzwe gifite filter ya Amicon 100.000 MWCO (Merck, UK), gikurwamo inshuro 20 hamwe na braffer, hanyuma cyongerwa kuri 25 ml. Mu gipimo cya DEAE Sepharose, hakoreshwa CV enye zifatanye na buffer mbere. Oza inkingi ukoresheje utubuto tune twa CV, hanyuma ushyiremo CV umunani ku murongo wa 0 kugeza 100 mM NaCl (muri buffer ihambiriye), hanyuma hagasigaramo CV enye zirimo 100 mM ihambiriye. Ibice bisigaye byashyizwemo sodium chloride hamwe n'ikigereranyo cya A880/A280 kiri hejuru ya 2.4 n'ikigereranyo cya A880/A805 kiri hejuru ya 4.6 byashyizwemo ~2 ml muri Amicon 100.000 MWCO centrifugal filter, hanyuma byuzuzwamo 1.5 CV IMAC mu gice cya mbere cya Buffer cya Superdex 200 16/600, hanyuma bishyirwamo muri buffer imwe hejuru ya 1.5 CV. Kusanya spectra zo kwinjizamo ibintu by’ibice bito bigabanya ingano hanyuma ushyiremo spectra zo kwinjizamo ibintu hamwe n’ibipimo bya A880/A280 hejuru ya 2.1 na A880/A805 hejuru ya 4.6 kugeza 100 A880, bihita bikoreshwa mu gutegura gridi ya TEM ikonje cyangwa bikabikwa kuri -80°C kugeza igihe bikenewe.
Icyuma gikonjesha cya Leica EM GP cyakoreshejwe mu gutegura imiyoboro ya TEM ifite ubushyuhe buke. Iyi complex yashyizwe muri IMAC buffer kuri A880 ya 50, hanyuma 5μl ishyirwa ku muyoboro mushya wa QUANTIFOIL 1.2/1.3 wa karuboni utwikiriwe n'urumuri (Agar Scientific, UK). Shyira icyuma kuri 20°C n'ubushyuhe bwa 60% mu gihe cy'amasegonda 30, hanyuma ugikureho cyumutse mu gihe cy'amasegonda 3, hanyuma ugizimye muri ethane y'amazi kuri -176°C.
Amakuru ya RC-LH114-W yafashwe kuri eBIC (Ikigo cy’Ubuhanga mu by’Ikoranabuhanga mu by’Indabyo) (British Diamond Light Source) hakoreshejwe mikorosikopi ya Titan Krios, ikora ku muvuduko wihuta wa 300kV, ifite ubushobozi bwo kongera ingufu bwa 130.000× n’ingufu za- Hitamo icyuho cya 20 eV. Gatan 968 GIF Quantum ifite detector ya K2 peak yakoreshejwe mu gufata amashusho mu buryo bwo kubara kugira ngo ikusanye amakuru. Ingano ya pikseli yagenzuwe ni 1.048Å, naho igipimo cya dose ni 3.83 e-Å-2s-1. Yakusanyije filime mu masegonda 11 ayigabanyamo ibice 40. Koresha agace gatwikiriwe na karubone kugira ngo wongere urebe mikorosikopi, hanyuma ukusanye filime eshatu kuri buri mwobo. Muri rusange, filime 3130 zakusanyijwe, zifite agaciro ka defocus kari hagati ya -1 na -3μm.
Amakuru y’ikigo cya RC-LH116 yakusanyijwe hakoreshejwe mikorosikopi imwe muri Laboratwari ya Asterbury Biostructure (Kaminuza ya Leeds, mu Bwongereza). Amakuru yakusanyijwe mu buryo bwo kubara hakoreshejwe uburyo bwo kongera uburebure bwa 130 k, kandi ingano ya pikseli yashyizwe kuri 1.065 Å hamwe n’umubare wa 4.6 e-Å-2s-1. Iyi filime yafashwe amajwi mu masegonda 12 igabanywamo ibice 48. Muri rusange, filime 3359 zakusanyijwe, zifite agaciro ka defocus kari hagati ya -1 na -3μm.
Gutunganya amakuru yose bikorwa muri Relion 3.0 (42). Koresha Motioncorr 2 (43) kugira ngo ukosore ingendo y'imirasire hakoreshejwe uburemere bw'ingano, hanyuma ukoreshe CTFFIND 4.1 (44) kugira ngo umenye parametre ya CTF (umurimo wo kohereza itandukaniro). Fotografiya zisanzwe nyuma y'ibi byiciro bya mbere byo gutunganya zigaragara ku Ishusho ya 2. S16. Icyitegererezo cyo guhitamo kidakora kiboneka mu guhitamo intoki pixel 250 z'uduce 1000 mu ishusho ya pixel 250 kandi nta cyiciro cy'ibice bibiri (2D) kigaragara, bityo bikabuza ibyiciro bihuye n'ibyanduye cyangwa bidafite imiterere igaragara. Hanyuma, guhitamo kidakora kidakora ku duce twose tw'amafotografi, maze RC-LH114-W yari uduce 849.359, naho RC-LH116 yari uduce 476.547. Uduce twose twatoranijwe twagiye mu byiciro bibiri bya 2D bitavugwaho rumwe, kandi nyuma ya buri gikorwa, utwo duce duhura n'agace ka karuboni, ubwandu bw'icyitegererezo, nta bintu bigaragara cyangwa uduce dufatanye cyane biranze, bigatuma utwo duce 772.033 (90.9%) na 359.678 (75.5%) dukoreshwa mu gushyira mu byiciro 3D bya RC-LH114-W na RC-LH116. Icyitegererezo cya mbere cya 3D cyakozwe hakoreshejwe uburyo bwa stochastic gradient descent. Hakoreshejwe icyitegererezo cya mbere nk'icyitegererezo, utwo duce twatoranijwe dushyirwa mu byiciro bine muri 3D. Hakoreshejwe icyitegererezo muri iki cyiciro nk'icyitegererezo, kora isesengura rya 3D ku duce two mu cyiciro kinini, hanyuma ukoreshe akayunguruzo ka mbere ka 15Å low-pass kugira ngo utwikire agace ka solvent, wongereho pixel 6 z'impande zoroshye, hanyuma ukoreshe pixels nyuma yo gutunganya pixels kugira ngo ukosore imikorere ya Gatan K2 peak Modulation transfer ya detector yo hejuru. Ku makuru ya RC-LH114-W, iyi moderi ya mbere yahinduwe hakurwaho ubucucike bukomeye ku nkengero za mask (itandukanyijwe n'ubucucike bw'ibanze muri UCSF Chimera). Modeli zavuyemo (ibipimo bya RC-LH114-W na RC-LH116 ni 3.91 na 4.16 Å, uko bikurikirana) zikoreshwa nk'icyitegererezo cy'icyiciro cya kabiri cy'ishyirwa mu byiciro bya 3D. Uduce twakoreshejwe dushyirwa mu cyiciro cya mbere cya 3D kandi ntabwo dufite isano ikomeye n'akarere. Bihura cyangwa nta miterere igaragara. Nyuma y'icyiciro cya kabiri cy'ishyirwa mu byiciro bya 3D, icyiciro gifite ubushobozi bwo hejuru cyatoranijwe [Kuri RC-LH114-W, icyiciro kimwe ni utuntu 377.703 (44.5%), kuri RC-LH116, hari ibyiciro bibiri, byose hamwe ni utuntu 260.752 (54.7%), Aho ari bimwe gusa iyo bihujwe nyuma yo kuzenguruka kw'ibanze hamwe n'itandukaniro rito]. Uduce twatoranijwe twongera gukurwa mu gasanduku ka pixel 400 hanyuma tugatunganywa hakoreshejwe uburyo bwa 3D. Agapfukamunwa gakozwe hakoreshejwe akayunguruzo ka mbere ka 15Å low-pass, kwagura ikarita ya pixel 3 na mask yoroshye ya pixel 3. Hakoreshejwe uburyo bwa CTF bwo gutunganya buri duce, gukosora ingendo za buri duce hamwe n'icyiciro cya kabiri cyo gutunganya buri duce, gutunganya 3D, gupfuka solvent no gutunganya nyuma y'intambwe bikorwa nyuma ya buri ntambwe kugira ngo harusheho kunozwa imiterere yavuyemo. Hakoreshejwe agaciro ka FSC (Fourier Shell Correlation Coefficient) ka 0.143, imyanzuro ya moderi za nyuma za RC-LH114-W na RC-LH116 ni 2.65 na 2.80Å, uko bikurikirana. Umurongo wa FSC w'icyitegererezo cya nyuma ugaragara ku Ishusho ya 2. S17.
Urutonde rwa poroteyine rwose rukurwa kuri 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); Poroteyine-W (PufW; UniProt ID: Q6N1K3). SWISS-MODEL (45) yakoreshejwe mu kubaka icyitegererezo cya homology cya RC, kirimo urutonde rwa poroteyine rwa RC-L, RC-M na RC-H kandi imiterere ya kristu ya Rba. sphaeroides RC yakoreshejwe nk'icyitegererezo (PDB ID: 5LSE) (46). Koresha igikoresho cya "fit map" muri UCSF Chimera kugira ngo uhuze icyitegererezo cyakozwe n'ikarita (47), unoze imiterere ya poroteyine, kandi ushyireho cofactor [4 × BChl a (izina rya monomer library residue = BCL), 2 × BPh a (BPH), ubwoko bumwe cyangwa bubiri bwa UQ10 (U10), icyuma kimwe kitari heme (Fe) n'ikindi cya 3,4-dihydrohexacarbonylcholine (QAK)] ukoreshe Coot (48) kugira ngo wongereho. Kubera ko QAK itaboneka muri monomer library, yashyizwe mu byiciro hakoreshejwe igikoresho cya eLBOW muri PHENIX (49).
Hanyuma, igice cya LH1 cyubatswe. Mbere na mbere, igikoresho cyo kubaka cyikora muri PHENIX (49) cyakoreshejwe mu gukora igice cya LH1 hakoreshejwe ikarita n'uruhererekane rwa poroteyine ya LH1-α na LH1-β nk'inyongera. Hitamo igice cya LH1 cyuzuye cyane, ukikuremo hanyuma ukishyire muri Coot, wongeremo uruhererekane rubura muri cyo, hanyuma utunganye imiterere yose n'intoki mbere yo kongeramo BCls ebyiri (BCL) na spirilloxanthin (CRT) [nk'uko bigaragazwa na Rps Ubucucike bwa LH1 complex n'ibikubiye muri carotenoid bizwi. Ubwoko (17)]. Kopera igice cya LH1 cyose, hanyuma ukoreshe "Docking Map Tool" ya UCSF Chimera kugira ngo ushyireho agace ka LH1 gaherereye hafi aho, hanyuma ugorore muri Coot; subiramo kugeza igihe ibice byose bya LH1 byubatswe. Ku miterere ya RC-LH114-W, binyuze mu gukuramo ubucucike butagabanijwe muri Coot, poroteyine igabanywa mu bice bisigaye bitari poroteyine muri USCF Chimera map maze igikoresho cya Autobuild gikoreshwa mu gushyiraho icyitegererezo cy’ibanze, hamwe n’ibice bisigaye (poroteyine-W) Modeling. Muri PHENIX (49). Ongeraho urutonde urwo arirwo rwose rubura kuri icyitegererezo cyabonetse muri Coot (48), hanyuma utunganye icyitegererezo cyose n’intoki. Ubucucike butagabanijwe bujyanye n’uruvange rwa lipide (PDB monomer library ID ya CDL = CDL, POPC = 6PL na POPG = PGT), β-DDM detergent (LMT) na UQ10 molecules (U10). Koresha PHENIX optimization (49) na optimization n’intoki muri Coot (48) kugira ngo unoze icyitegererezo cy’ibanze cyose kugeza igihe imibare y’icyitegererezo n’ubwiza bw’ishusho y’uburyo ikwiranye bidashobora kunozwa kurushaho. Amaherezo, koresha LocScale (50) kugira ngo ukarishe ikarita yo mu gace, hanyuma ukore izindi ngendo nyinshi zo kwerekana ubucucike butagabanijwe no gutunganya mu buryo bwikora no mu buryo bw’intoki.
Peptide, cofactors n'izindi lipide na quinones byashyizwe mu bucucike bwabyo bigaragara ku Ishusho ya 1 na 2. Kuva kuri S18 kugeza kuri S23. Amakuru y'imibare y'icyitegererezo cya nyuma agaragara ku Imbonerahamwe ya S1.
Keretse iyo byavuzwe ukundi, spectra zo gufata UV/Vis/NIR zakusanyijwe kuri spectrophotometer ya Cary60 (Agilent, USA) ku ntera ya 1 nm kuva kuri 250 nm kugeza 1000 nm n'igihe cyo guhuza cya 0.1s.
Shyira icyitegererezo muri cuvette ya quartz ifite inzira ya mm 2 kuri A880 ya 1, hanyuma ukuremo spectrum y'ubushyuhe iri hagati ya nm 400 na 1000. Spectrum z'uruziga za dichroic zakusanyijwe kuri Jasco 810 spectropolarimeter (Jasco, Ubuyapani) ku ntera ya nm 1 hagati ya nm 400 na 950 ku gipimo cya scan cya 20 nm min-1.
Igipimo cyo kuzimira kw'umukara kigenwa no kugabanya imiterere y'ibanze kuri A880 ya hafi 50. Kuramo ingano ya 10μl muri 990μl binding buffer cyangwa methanol, hanyuma uhuze spectrum yo kuzimira ako kanya kugira ngo ugabanye kwangirika kwa BChl. Ingano ya BChl muri buri ngero ya methanol yabazwe hakoreshejwe 771 nm ya 54.8 mM-1 cm-1, hanyuma ikigereranyo cyo kuzimira cyagenwe (51). Gabanya igipimo cya BChl cyapimwe na 32 (RC-LH114-W) cyangwa 36 (RC-LH116) kugira ngo hamenyekane igipimo cy'ingenzi cy'ingenzi, hanyuma kigakoreshwa mu kumenya spectrum yo kuzimira y'icyitegererezo kimwe cyakusanyijwe muri ikigereranyo cyo kuzimira cy'umukara. Ibipimo bitatu byafashwe kuri buri ngero, kandi ikigereranyo cyo kuzimira cya BChl Qy maximum cyakoreshejwe mu kubara. Igipimo cyo kuzimira cya RC-LH114-W gipimwe kuri 878 nm ni 3280±140 mM-1 cm-1, mu gihe igipimo cyo kuzimira cya RC-LH116 gipimwe kuri 880 nm ari 3800±30 mM-1 cm-1.
UQ10 yapimwe hakurikijwe uburyo buri muri (52). Muri make, HPLC (RP-HPLC) yakozwe hakoreshejwe sisitemu ya Agilent 1200 HPLC. Shyiraho 0.02 nmol ya RC-LH116 cyangwa RC-LH114-W muri 50μl ya 50:50 methanol:chloroform irimo 0.02% (w/v) ferric chloride, hanyuma ushyiremo Beckman Coulter Ultrasphere ODS 4.6 mm. Shyiraho muri 1 ml-1 min-1 kuri 40°C muri solvent ya HPLC (80:20 methanol:2-propanol) ku nkingi ya ×25 cm. Kora isocratic elution muri solvent ya HPLC kugira ngo urebe niba imyuka iri kuri 275 nm (UQ10), 450 nm (carotenoids) na 780 nm (BChl) mu gihe cy'isaha 1. Agace k’umurambararo ka chromatogramu ya 275 nm mu minota 25.5 kari kashyizwe hamwe, katari karimo ibindi bintu bishobora kugaragara. Agace k’umurambararo gakoreshwa mu kubara ingano ya molar ya UQ10 yakuwemo hakurikijwe umurongo wa calibration wabazwe uhereye ku gutera ibipimo by’ubuziranenge kuva kuri 0 kugeza kuri 5.8 nmol (Ishusho S14). Buri rugero rwasesenguwe mu buryo butatu, kandi ikosa ryatangajwe rihuye na SD y’ikigereranyo.
Hateguwe umuti urimo RC-LH1 complex ifite ubushobozi bwo kwinjiza amazi bwa Qy bwa 0.1 hamwe na 30 μM yagabanutseho cytochrome c2 y'umutima w'ifarashi (Merck, UK) na 0 kugeza kuri 50 μMUQ2 (Merck, UK). Ingero eshatu za mililitiro 1 zateguwe kuri buri gipimo cya UQ2 maze zishyirwa mu mwijima ijoro ryose kuri 4°C kugira ngo hamenyekane neza ko zihuye neza n'umwijima mbere yo gupimwa. Umuti washyizwe muri OLIS RSM1000 modular spectrophotometer ifite 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm hagati na 0.6 mm outlet slits. Akayunguruzo k'uburebure bwa 600 nm gashyirwa ku muryango w'agace k'ifoto y'icyitegererezo n'agace k'ifoto y'icyitegererezo kugira ngo hatabaho urumuri rwo gushyuha. Ubushobozi bwo kwinjiza amazi bwagenzuwe kuri 550 nm hamwe n'igihe cyo guhuza 0.15 s. Urumuri rwo gushyushya rusohoka kuri LED ya 880 nm M880F2 (Light Emitting Diode) (Thorlabs Ltd., UK) binyuze mu mugozi wa fiber optique ku bunini bwa 90% binyuze muri DC2200 controller (Thorlabs Ltd., UK) kandi rwoherezwa ku isoko ry'urumuri ku nguni ya 90° ya. Umurabyo upimye utandukanye n'indorerwamo kugira ngo ugarure urumuri urwo ari rwo rwose rutari rwakwinjizwa n'icyitegererezo bwa mbere. Reba uburyo bwo kwinjiza urumuri iminota 10 mbere y'uko urumuri rumurika iminota 50. Hanyuma uburyo bwo kwinjiza urumuri bwakurikiranwe mu gihe cy'iminota 60 mu mwijima kugira ngo harebwe uburyo quinolol igabanya cytochrome c23 + mu buryo butunguranye (reba Ishusho S8 kugira ngo umenye amakuru y'ibanze).
Amakuru yatunganyijwe hakoreshejwe uburyo bwo gushyira igipimo cy’ibanze mu masegonda 0.5 kugeza 10 (bitewe n’igipimo cya UQ2) no gushyira impuzandengo y’ibipimo bitatu kuri buri gipimo cya UQ2. Igipimo cya RC-LH1 cyabazwe hakurikijwe igipimo cyo kuzimira cyakoreshejwe mu guhindura igipimo mu buryo bwo gukamura, cyashushanyijwe muri Origin Pro 2019 (OriginLab, USA), maze gishyirwa muri model ya Michaelis-Menten kugira ngo hamenyekane agaciro ka Km na Kcat kagaragara.
Ku bipimo by’igihe gito byo kwinjiza, icyitegererezo cya RC-LH1 cyavanzwe kugeza kuri ~2μM muri IMAC buffer irimo 50 mM sodium ascorbate (Merck, USA) na 0.4 mM Terbutin (Merck, USA). Aside ascorbic ikoreshwa nk'umutanga electron witangira, naho tert-butaclofen ikoreshwa nk'umuti ubuza QB kugira ngo utanga RC nyamukuru akomeze kugabanuka (ni ukuvuga, atari fotooxidized) mu gihe cyose cyo gupima. Hafi mililitiro 3 z'icyitegererezo zongerwa mu karemangingo gasanzwe gazunguruka (hafi metero 0.1 z'umurambararo, 350 RPM) gafite uburebure bwa mm 2 bw'inzira y'urumuri kugira ngo umenye neza ko icyitegererezo kiri mu nzira ya laser gifite igihe gihagije cyo guhuza umwijima hagati y'imivuduko yo gushyushya. Koresha imivuduko ya laser ya ~100-fs kugira ngo wongere uburyo bwa laser ya Ti: Sapphire (Spectra Physics, USA) kugira ngo wongere icyitegererezo kuri 880 nm ku gipimo cyo gusubiramo cya 1 kHz (20 nJ kuri NIR cyangwa 100 nJ kuri Vis). Mbere yo gukusanya amakuru, shyira icyitegererezo mu rumuri rushyushye mu gihe cy'iminota 30. Kugaragara bizatera QA kudakora (bishobora kugabanya QA rimwe cyangwa kabiri). Ariko nyamuneka menya ko iki gikorwa gishobora gusubizwa inyuma kuko nyuma y'igihe kirekire cyo guhinduka mu mwijima, RC izagaruka buhoro buhoro ku bikorwa bya QA. Spektrometeri ya Helios (Ultrafast Systems, USA) yakoreshejwe mu gupima spectra y'igihe gito ifite igihe cyo gutinda kuva kuri -10 kugeza kuri 7000 ps​​. Koresha porogaramu ya Surface Xplorer (Ultrafast Systems, USA) kugira ngo uvane amakuru mu matsinda, hanyuma uvange hanyuma ushyireho uburyo busanzwe. Koresha porogaramu ya CarpetView (Light Conversion Ltd., Lituwaniya) kugira ngo ukoreshe amakuru ahujwe kugira ngo ubone spectra zitandukanye zijyanye no kubora, cyangwa ukoreshe imikorere ihuza exponents nyinshi hamwe n'igisubizo cy'igikoresho kugira ngo gihuze n'ihindagurika rya spectra y'uburebure bumwe mu ntangiriro (OriginLab, USA).
Nkuko byavuzwe haruguru (53), hateguwe filime ya fotosynthetic irimo LH1 complex idafite RC na antene ya LH2 yo mu gice cya peripheral. Urwo rurerure rwavanzwe muri tris 20 mM (pH 8.0) hanyuma rushyirwa muri cuvette ya quartz ifite inzira ya optique ya mm 2. Ingufu ya laser ya 30nJ yakoreshejwe kugira ngo ishyushye icyitegererezo kuri 540 nm hamwe n'igihe cyo gutinda kuva kuri -10 kugeza kuri 7000 ps. Gutunganya amakuru nk'uko byasobanuwe kuri Rps. pal sample.
Uru rusobe rw'amazi rwashyizwemo amazi hakoreshejwe centrifugation kuri 150.000 RCF mu gihe cy'amasaha 2 kuri 4°C, hanyuma ubwiyongere bwarwo kuri 880 nm bwongera gushyirwa muri 20 mM tris-HCl (pH 8.0) na 200 mM NaCl. Shyira urusobe mu kuvanga buhoro buhoro 2% (w/v) β-DDM mu gihe cy'isaha 1 mu mwijima kuri 4°C. Icyitegererezo cyavanzwe muri 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) kugeza ku gipimo cya poroteyine cya 2.5 mg ml-1 (Bio-Rad analysis). Hakozwe ubundi buryo bwo gutunganya hakoreshejwe uburyo bwatangajwe mbere (54), hatangirana no kuvanga 50 μg poroteyine muri 50 μl TEAB irimo 1% (w/v) sodium laurate (Merck, UK). Nyuma yo kuyikoresha mu gihe cy'amasegonda 60, yagabanijwe hakoreshejwe tris (2-carboxyethyl) fosfini ya 5 mM (Merck, UK) kuri 37°C mu gihe cy'iminota 30. Kuri S-alkylation, shyiramo icyitegererezo hamwe na 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) hanyuma wongereho uvuye muri isopropanol stock solution ya 200 mM mu gihe cy'iminota 10 ku bushyuhe bw'icyumba. Igogorwa rya Proteolytic ryakozwe hongerwamo 2 μg trypsin / endoproteinase Lys-C mix (Promega UK) hanyuma rishyirwa kuri 37°C mu gihe cy'amasaha 3. Laurate surfactant yakuwemo hongerwamo 50 μl ethyl acetate na 10 μl 10% (v / v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) hanyuma vortexing mu gihe cy'amasegonda 60. Gutandukanya ibice byazamuwe binyuze mu gupima amashanyarazi kuri 15.700 RCF mu gihe cy'iminota 5. Dukurikije amabwiriza y'uwakoze, inkingi ya C18 spin (Thermo Fisher Scientific, UK) yakoreshejwe mu gusohora no gukuramo umunyu witonze mu gice cyo hasi kirimo peptide. Nyuma yo kuma hakoreshejwe gupima amashanyarazi, icyitegererezo cyashongeshejwe muri 0.5% TFA na 3% acetonitrile, maze 500 ng yasesengurwa hakoreshejwe nanoflow RP chromatography hamwe na mass spectrometry hakoreshejwe ibipimo bya sisitemu byasobanuwe mbere.
Koresha MaxQuant v.1.5.3.30 (56) kugira ngo umenye poroteyine kandi uyipime kugira ngo ushakishe Rps. palustris proteome database (www.uniprot.org/proteomes/UP000001426). Amakuru ya proteomics ya spectrometry yashyizwe muri ProteomeXchange Alliance binyuze mu bubiko bw'abafatanyabikorwa ba PRIDE (http://proteomecentral.proteomexchange.org) munsi y'ikimenyetso cy'amakuru PXD020402.
Kugira ngo hakorwe isesengura rya RPLC hamwe na electrospray ionization mass spectrometry, RC-LH1 complex yakozwe muri wild-type Rps. Hakoreshejwe uburyo bwatangajwe mbere (16), ingano ya poroteyine yakozwe mu turemangingo twa palustris yari 2 mg-1 muri 20 mM Hepes (pH 7.8), 100 mM NaCl na 0.03% (w/v) β- (Bio-Rad analysis)) DDM. Dukurikije amabwiriza y'uwakoze, koresha 2D purification kit (GE Healthcare, USA) kugira ngo ukuremo 10 μg poroteyine hakoreshejwe uburyo bwo kuvura, hanyuma ushongeshe precipitate muri 20 μl 60% (v / v) aside formic (FA), 20% (v / v) Acetonitrile na 20% (v / v) amazi. Mikorolitiro eshanu zasesenguwe na RPLC (Dionex RSLC) hamwe na mass spectrometry (Maxis UHR-TOF, Bruker). Koresha inkingi ya MabPac 1.2×100 mm (Thermo Fisher Scientific, UK) kugira ngo utandukanye kuri 60°C na 100μlmin -1, hamwe n'umuvuduko wa 85% (v / v) solvent A [0.1% (v / v) FA na 0.02% (V / v) TFA aqueous solution] kuri 85% (v / v) solvent B [0.1% (v / v) FA na 0.02% (v / v) muri 90% (v / v) acetonitrile TFA] Ukoresheje electrospray isanzwe ionization source na default parameters mu gihe kirenga iminota 60, spectrometer y'uburemere ibona 100 kugeza 2750 m/z (mass-to-charge ratio). Ukoresheje urubuga rwa ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), shyira spectrometer y'uburemere ku duce duto tw'iki kigo.
Uturemangingo twakuze amasaha 72 munsi ya 100 ml NF-low (10μMm-2 s-1), iri hagati (30μMm-2 s-1) cyangwa iri hejuru (300μMm-2 s-1). Uturemangingo twa M22 (M22 medium aho ammonium sulfate ivamo hanyuma sodium succinate igasimburwa na sodium acetate) mu icupa rya 100 ml screw-top (23). Mu minota itanu ya 30, amasaro y'ikirahure ya mikoroni 0.1 yashyizwemo amasaro ku gipimo cya 1:1 kugira ngo atunganye uturemangingo maze akonjeshwa ku rubura mu gihe cy'iminota 5. Ibintu bidashongesha, uturemangingo tutavunitse n'amasaro y'ikirahure byakuweho hakoreshejwe centrifugation kuri 16.000 RCF mu gihe cy'iminota 10 muri microcentrifuge ya benchtop. Ururenda rwatandukanyijwe muri rotor ya Ti 70.1 ifite RCF 100.000 muri 20 mM tris-HCl (pH 8.0) hamwe n'umuvuduko wa sucrose wa 40/15% (w/w) mu gihe cy'amasaha 10.
Nkuko byasobanuwe mu kazi kacu ka mbere, gusuzuma ubudahangarwa bw'umubiri bwa His tag kuri PufW (16). Muri make, complex ya purified core (11.8 nM) cyangwa membrane irimo ingano imwe ya RC (igenwa no oxidation ikuraho spectrum yagabanijweho y'itandukaniro no guhuza umutwaro kuri gel yanduye) muri 2x SDS loading buffer (Merck, UK) yagabanijwe kabiri. Poroteyine zatandukanyijwe kuri gel ya 12% bis-tris NuPage (Thermo Fisher Scientific, UK). Gel yasizwe ibara rya Coomassie Brilliant Blue (Bio-Rad, UK) kugira ngo ishyiremo kandi igaragaze agace gato ka RC-L. Poroteyine kuri gel ya kabiri yimuwe muri membrane ya polyvinylidene fluoride (PVDF) yakoreshejwe na methanol (Thermo Fisher Scientific, UK) kugira ngo ikoreshwe mu budahangarwa bw'umubiri. Ururenda rwa PVDF rwafunzwe muri 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 na 5% (w / v) ifu y'amata yakuwemo amavuta, hanyuma rushyirwamo anti-His primary antibody (muri Dilute antibody buffer [50 mM tris-HCl (pH 7.6), 150 mM NaCl na 0.05% (v / v) Tween-20] muri 1:1000 A190-114A, Bethyl Laboratories, USA) mu gihe cy'amasaha 4. Nyuma yo gukaraba inshuro 3 mu minota 5 mu gice cy’ubutabazi bw’umubiri, agace k’ubutabazi kahujwe na horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (yavanzwemo 1:10.000 mu gice cy’ubutabazi bw’umubiri) Shyiramo kugira ngo hamenyekane (iminota 5 nyuma yo gukaraba inshuro 3 mu gice cy’ubutabazi bw’umubiri) hakoreshejwe WESTAR ETA C 2.0 chemiluminescence substrate (Cyanagen, Ubutaliyani) na Amersham Imager 600 (GE Healthcare, UK).
Mu gushushanya ubukana bwa buri gel irangi cyangwa inzira y’ubudahangarwa bw’umubiri, guhuza agace kari munsi y’umusozo no kubara ubukana bwa RC-L (gel irangi) na Poroteyine-W (isuzuma ry’ubudahangarwa bw’umubiri), muri ImageJ (57) Tegura ishusho. Ibi bipimo byahinduwe molar ratios hashingiwe ku kuba igipimo cya RC-L na poroteyine-W mu gipimo cya RC-LH114-W cyonyine cyari 1:1 no guhuza amakuru yose uko yakabaye.
Ku bindi bikoresho by'inyongera kuri iyi nkuru, reba 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
Imiterere ifite ubushobozi bwo hejuru bw'urumuri rwa trap 1 mu kigo cy'ibizamini itanga ubumenyi bushya ku mikorere ya 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
Imiterere ifite ubushobozi bwo hejuru bw'urumuri rwa trap 1 mu kigo cy'ibizamini itanga ubumenyi bushya ku mikorere ya quinone.
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