Mu kiseera kino†Endagiriro eriwo kati: OX11 0DE, Bungereza, Ekizimbe kya Dayimanda, Harwell Sayansi n’Obuyiiya Park, Dietcote, Oxfordshire, Bungereza, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Ekizibu ky’okukungula ekitangaala mu kifo ky’ensengekera 1 (RC-LH1) kye kitundu ekikulu eky’okusengejja ekitangaala ekya bakitiriya abakola ekitangaala ekya kakobe. Twaleeta ensengekera bbiri eza cryo-electron microscopy ez’ekizibu kya RC-LH1 okuva mu Rhodopseudomonas palustris. Ensengekera ya 2.65-Å resolution structure ya RC-LH114-W complex erimu 14 subunit LH1 loops ezeetoolodde RC, ezitaataaganyizibwa protein W, ate complex etali protein-W ye RC composition yonna eyeetooloddwa RC. Yaggaddwa 16 subunit LH1 loopu. Okugerageranya ensengekera zino kuwa amagezi ku nkyukakyuka ya quinone mu RC-LH1 complex, omuli enkyukakyuka z’ensengekera ezaali tezimanyiddwa emabegako nga basiba quinone mu kifo kya RC QB, awamu n’ekifo ky’ebifo ebiyambako ebisiba quinone, ebiyamba Okuziyisa mu RC. Ensengekera ey’enjawulo eya puloteyina ya W eremesa okuggalawo loopu ya LH1, bwe kityo ne kikola omukutu ogw’okwanguyiza okuwanyisiganya kwa quinone/quinolone.
Amasoboza agaweebwa okusengejja ekitangaala gasobola okuyimirizaawo kumpi ebiramu byonna ku nsi, era galina obusobozi bungi mu tekinologiya w’ebiramu eby’enjuba. Wadde nga zitumbula okusengejja ekitangaala mu nsi yonna, obuwuka obukola ekitangaala obwa kakobe era bulaga engeri z’amasoboza ez’enjawulo n’obusobozi bw’okukyusakyusa ebiriisa. Zisobola okwewala okuyungibwa kw’ekitangaala ne zikula nga bakitiriya ezitali za njawulo mu nzikiza, zisobola okutereeza nayitrojeni ne kaboni dayokisayidi, okukola haidrojeni, n’okusaanyaawo ebirungo ebiwunya (1-3). Okusobola okuwa amasoboza eri enkola zino, ekitangaala kirina okukyusibwa amangu era mu ngeri ennungi okufuuka amasoboza ag’eddagala. Enkola eno etandika nga ekizibu kya antenna ekikwata ekitangaala kinyiga ekitangaala ne kikyusa amasoboza agasibiddwa mu kifo ky’ensengekera (RC), bwe kityo ne kitandika okwawukana kwa chajingi (4 – 7). Ekitundu ekikulu eky’okusengejja ekitangaala mu bakitiriya ezikola ekitangaala eky’erangi ya kakobe kikolebwa ekika kya 2 RC, nga kyetooloddwa ekizibu ky’okukungula ekitangaala 1 (LH1), ekikola ekizibu ky’omusingi gwa RC-LH1. LH1 ekolebwa ensengekera ya αβ heterodimers ezikoona, buli emu ku zo esiba molekyu bbiri eza bacterial chlorophyll (BChl) a ne carotenoids emu oba bbiri (8-12). Antenna ya LH1 esinga okuba ennyangu erimu αβ heterodimers 16 oba 17 ezeetoolodde RC (9-13) mu loopu enzigale, naye mu bizibu ebirala ebikulu, peptides eziyita mu lususu zisalako okugenda mu maaso kwa LH1 eyeetoolodde , Bwe kityo ne zitumbula okusaasaana kwa quinol/quinone wakati wa RC ne cytochrome bc1 complex (11, 13-15). Ekimera eky’ekika kya purple phototrophic Rhodopseudomonas (Rps.) kiramu kya muze ekisobola okutegeera okutambuza kw’amasoboza n’obusannyalazo okuwagira okusengejja ekitangaala. Ensengekera ya kirisitaalo esooka eya Rps. Omuze gwa palustris RC-LH1 complex ye RC, eyeetooloddwa 15 heterodimeric LH1 loops, ezitaataaganyizibwa puloteyina etamanyiddwa eyitibwa “Protein W” ( 14 ). Oluvannyuma puloteyina-W yazuulibwa nga RPA4402, nga eno puloteyina ya 10.5kDa etamanyiddwa ng’erina helices ssatu eziteeberezebwa okuyita mu lususu (TMH) ( 16 ). Tuteesa okukyusa erinnya ly’obuzaale bwa rpa4402 obuwandiika puloteyina W okudda ku pufW okusobola okukwatagana n’amannya agakozesebwa ku buzaale obuwandiika RC-L, M (pufL, pufM) ne LH1α, β (pufA, pufB) subunits. Ekyewuunyisa, protein-W eri mu bitundu nga 10% byokka ebya RC-LH1, ekiraga Rps. palustris ekola ebizibu bibiri eby’enjawulo ebya RC-LH1. Wano, tutegeeza ensengekera za cryo-EM (cryo-EM) ez’obulungi obw’amaanyi ez’ebizibu bibiri ebikulu, ekimu nga kiriko puloteyina W ne 14 αβ heterodimers, ekirala nga tekirina puloteyina W n’olukoba lwa 16 Heterodimer LH1 oluggaddwa. Ensengekera yaffe ekiikirira enkyukakyuka y’omutendera mu kutegeera ekizibu kya RC-LH1 ekya Rps. palustris, kubanga twekenneenyezza omuwendo gw’abantu ogumu ogwa buli nkyukakyuka era tulina okusalawo okumala okugabanya obulungi buli peptide ne langi ezisibiddwa n’amasavu ne quinones ezikwatagana. Okugerageranya ensengekera zino kulaga nti puloteyina za TMH essatu-W ezitannazuulibwa mu kizibu ekirala kyonna ekya RC-LH1 okutuusa kati zikola omukutu gwa quinone okwanguya okuwanyisiganya kwa quinone/quinolone. Ebifo ebiwerako ebikuumibwa ebisiba lipid ne quinone bizuuliddwa, era tulaze enkyukakyuka empya mu nsengekera oluvannyuma lw’okugatta quinone ne RC, ekiyinza okuba nga kituukirawo ku photosystem II (PSII) RC y’ebiramu ebirina oxygenated phototrophic. Ebizuuliddwa byaffe biwa amagezi amapya ku kinetics y’okusiba quinone/quinolone n’okuwanyisiganya mu RC-LH1 core complex ya purple phototrophic bacteria.
Okusobola okwanguyiza okunoonyereza mu bujjuvu ku bizibu ebibiri ebisangibwa mu Rps. palustris, twawula buli RC-LH1 nga tukozesa enkola z’ebiramu. Ekizibu kya puloteyina W ekibulamu (wano kiyitibwa ΔpufW) kyalongoosebwa okuva mu kika ekitaliimu ggiini ya pufW ( 16 ), era ekizibu kya RC-LH1 kimu kyokka kye kisobola okukolebwa. Ekizibu ekirimu puloteyina W kikolebwa ekika. Puloteeni W ey’ekika kino ekyusiddwa n’akabonero ka His aka 10x ku C-terminus yaayo, olwo ekizibu ekirimu puloteyina W ne kisobola okugattibwa obulungi ne puloteyina W ezisinga obutaba nga kiziyiza ekyuma. Ekizibu kino kyawulwa bulungi (16) Affinity Chromatography (IMAC).
Nga bwe kiragibwa mu kifaananyi 1, ebizibu byombi birimu RC ya sub-unit ssatu (RC-L, RC-M ne RC-H) eyeetooloddwa antenna ya LH1. Ensengekera ya 2.80-A ey’ekizibu ekibulamu puloteyina-W eraga αβ heterodimers 16, nga zikola loopu ya LH1 enzigale eyeetoolodde ddala RC, mu kiseera kino eyitibwa ekizibu kya RC-LH116. Ensengekera ya 2.65Å ey’ekizibu ekirimu puloteyina-W erina 14-heterodimer LH1 esaliddwako puloteyina-W, wano eyitibwa RC-LH114-W.
(A ne B) Okukiikirira kungulu kw’ekirungo. (C ne D) Langi eziyungiddwa eziragibwa mu miggo. (E ne F) Ebizibu ebitunuuliddwa okuva ku ngulu wa cytoplasmic birina peptides ne LH1 subunits ezikiikirirwa mu katuni, era ziweebwa ennamba mu ssaawa okuva ku kituli kya protein-W [ekikwatagana n’ennamba ya Rba. ebizimba ebiyitibwa sphaeroides complex (13)]. Ku LH1-α, langi y’ekitundu kya puloteyina eba ya kyenvu; ku LH1-β, langi y’ekitundu kya puloteyina ya bbululu; ku puloteyina-W, puloteyina eba emmyufu; ku RC-H, kiba kya cyan; ku RC-L, ya Orange; ku RC-M, langi ya magenta. Cofactors zikiikirira emiggo, green ekiikirira molekyu za BChl ne BPh a, purple ekiikirira carotenoids, ate kiragala ekiikirira molekyu za UQ10. (G ne H) Okulaba okukuzibwa kw’ekituli kya puloteyina-W mu kitundu ekyenkanankana ekya RC-LH114-W complex (G) ne RC-LH116 complex (H). Cofactors ziragibwa mu ngeri y’okujjuza ekifo, chelated quinone eragiddwa mu bbululu. Ekituli kya puloteyina-W kiragibwa layini ya bbululu ennyiriri mu (G), ate obutuli obutono quinone/quinolol mw’esaasaana ku mpeta ya LH116 bulagibwa layini enkokola enjeru mu (H).
Ekifaananyi 1 (A ne B) kiraga RC eyeetooloddwa ensengekera eziggule oba eziggaddwa eza LH1αβ heterodimers, buli emu ku zo esiba BChl bbiri ne carotenoid emu (Ekifaananyi 1, C ne D). Okunoonyereza okwakolebwa emabegako kwalaga nti Rps ye LH1 complex. Mu kkubo ly’ebiramu erya spirulina xanthin, ebika bino birimu ebika bya carotenoids ebitabuddwa ( 17 ). Wabula spiropyrroxanthin ye carotenoid esinga era density yaayo ematiza. N’olwekyo, twasalawo okukola model ya spiroxanthin mu bifo byonna ebisiba LH1. Alpha ne beta polypeptides zibeera TMH emu nga zirina ebitundu eby’ebweru eby’olususu ebimpi (Ekifaananyi 1, A, B, E, ne F). Newankubadde nga density y’ebisigadde 17 ku C-terminus teyalabibwa, alpha polypeptide yakutulwa okuva ku Met1 okutuuka ku Ala46 mu complexes zombi. β polypeptide yakendeezebwa okuva ku Gly4 okutuuka ku Tyr52 mu RC-LH116, n’okuva ku Ser5 okutuuka ku Tyr52 mu RC-LH114-W. Tewali density ya 3 oba 4 N-terminal oba 13 C-terminal residues yalabibwa (Figure S1). Okwekenenya kwa mass spectrometry ku mixed RC-LH1 complex eyategekebwa okuva mu wild-type strain kwalaga nti ekitundu ekyabula kyali kiva ku heterologous cleavage ya peptides zino (Figure S1 ne S2). N-terminal formylation ya α-Met1 nayo yalabibwa (f). Okwekenenya kwalaga nti α-peptide erimu ebisigadde fMet1 okutuuka ku Asp42/Ala46/Ala47/Ala50, ate β-peptide erimu ebisigadde Ser2 okutuuka ku Ala53, ekikwatagana obulungi ne maapu ya EM density ey’ebbugumu eri wansi.
Okukwatagana kwa α-His29 ne β-His36 kufuula BChls maaso ku maaso; buli αβ heterodimer ekuŋŋaana ne baliraanwa baayo okukola olukoba oluggule (RC-LH114-W) oba olukoba oluggaddwa (RC-LH116) okwetoloola RC The exciton coupled pigment array (Figure 1, C and D). Bw’ogeraageranya ne 877 nm band ya RC-LH114-W, 880 nm absorption red shift ya RC-LH116 eri 3 nm (Ekifaananyi 2A). Naye, ensengekera ya circular dichroism kumpi y’emu (Ekifaananyi 2B), ekiraga nti wadde nga waliwo enjawulo entegeerekeka wakati wa loopu eziggule n’eziggaddwa, embeera y’ekitundu eya BChls efaanagana nnyo. Okukyukakyuka okumyufu okw’okunyiga kuyinza okuba nga kuva ku kukendeera kw’entambula y’ebbugumu n’okweyongera okutebenkera ku loopu enzigale (18, 19), enkyukakyuka mu kuyungibwa kwa langi okuva ku loopu enzigale (20, 21), oba okugatta ebikolwa bino ebibiri (11).
(A) Ultraviolet/visible/near-infrared absorption spectrum, entikko zazo ziteekeddwako akabonero ne langi zazo ezikwatagana era ne zifuuka normalized okutuuka ku ntikko ya BPh ku 775 nm. (B) Ensengekera ya dichroism ey’enkulungo eya normalized okutuuka ku BChl absorbance ku 805 nm. (C ne D) Ebifaananyi bya ΔA ebirondeddwa okuva mu bifaananyi by’okunyiga ebigonjoddwa mu kiseera ebya RC-LH114-W complex (C) ne RC-LH116 complex (D). Okusobola okugeraageranya obulungi, spektira zonna zikyusibwa okutuuka ku ∆A ya −A ku 0.2 ps. (E) Omutindo gwa cytochrome c2 oxidation oluvannyuma lw’okubunyisa obusannyalazo mu kubeerawo kw’ebirungo eby’enjawulo ebya UQ2 (laba ekifaananyi S8 ku data embisi). (F) Mu butoffaali obukuliddwa wansi w’ekitangaala eky’amaanyi amatono, aga wakati oba aga waggulu (10, 30 oba 300μMm-2 s-1, okusinziira ku), ebitundu bya puloteyina W ne RC-L mu kizibu ekirongooseddwa n’omugerageranyo gw’olususu olwawuddwamu. Laba omutindo gwa puloteyina nga okozesa SDS-polyacrylamide gel electrophoresis ne immunoassay (laba ekifaananyi S9 okufuna data embisi). Laga omugerageranyo ogukwatagana n’ekizibu kya RC-LH114-W ekirongooseddwa. Omugerageranyo gwa stoichiometric ogwa RC-L ne protein-W y’ekizibu kiri 1:1.
BChls eziri mu kifo 1 mu loopu ya αβ14 ekyukakyuka eya RC-LH114-W (Ekifaananyi 1, A, C, ne E) ziri kumpi n’omugabi wa RC omukulu (P) ne 6.8Å okusinga BChls ezenkanankana mu RC-LH116 (Ekifaananyi. 1, B, D, ne F, n’Ekifaananyi S3); wabula, ensengekera y’okunyiga ey’ekiseera (transient absorption kinetics) ey’ebizibu ebibiri eraga nti ku RC-LH114-W ne RC-LH116, enkyukakyuka z’obudde bw’okutambuza amasoboza ag’okusikirizibwa okuva ku LH1 okutuuka ku RC ziri 40 ±4 ne 44±3 ps (Ekifaananyi 2). , C ne D, Ekifaananyi S4 n’Ekipande S2). Era tewali njawulo nnene mu kukyusa mu byuma bikalimagezi munda mu RC (Ekifaananyi S5 n’ebiwandiiko eby’okugatta ebikwatagana). Tuteebereza nti okukwatagana okumpi okw’ekiseera ky’okutambuza amasoboza wakati wa LH1 ne RC-P kuva ku bbanga, enkoona n’amasoboza agayinza okufaanagana aga BChl ezisinga obungi mu loopu ebbiri eza LH1. Kirabika okunoonyereza ku nkola y’amasoboza ga LH1 okutuuka ku bbanga erisinga obutono si kyangu okusinga okutambuza amasoboza obutereevu okuva mu bifo ebitali birungi okutuuka ku RC. Luupu ya LH1 ey’olukoba oluggule mu RC-LH114-W nayo eyinza okuyita mu ntambula y’ebbugumu etali nnene wansi w’embeera z’ebbugumu eri wansi okusobola okwekenneenya enzimba, era waliwo ensengekera y’empeta ya αβ14 empanvu ku bbugumu erya bulijjo okuva ku bbanga lya langi ya βBChls mu kifo kya RC 1.
Ekizibu kya RC-LH116 kirimu BChls 32 ne carotenoids 16, era enteekateeka yaakyo okutwalira awamu y’emu n’eyo efunibwa okuva mu Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 ekika ( PDB ID 7C9R) (12) ne green ebiwuka ebiyitibwa algae (Blc.viridis) (PDB ID 6ET5) (10). Oluvannyuma lw’okukwatagana, okukyama okutono kwokka mu bifo bya αβ heterodimers kwe kwalabiddwa naddala 1-5, 15, ne 16 (Ekifaananyi S6). Okubeerawo kwa puloteyina-W kulina kinene kye kukola ku nsengekera ya LH1. TMH zaayo essatu ziyungiddwa ku loopu ennyimpi, nga N-terminal eri ku ludda lwa lumen olwa complex ate C-terminal eri ku ludda lwa cytoplasmic (Figures 1A and 3, A to D). Protein-W okusinga tekwata mazzi (Figure 3B), era TMH2 ne TMH3 zikwatagana ne LH1αβ-14 okukola transmembrane surface (Ekifaananyi 3, B ne E okutuuka ku G). Enkolagana esinga kukolebwa bisigalira bya Phe, Leu ne Val mu kitundu ekiyita mu lususu. Ebisigadde bino bitumbibwamu amino asidi ezitawunya mazzi ne langi za αβ-14. Ebimu ku bisigadde eby’enjuba (polar residues) nabyo biyamba mu nkolagana, omuli n’enkolagana ya haidrojeni wakati wa W-Thr68 ne β-Trp42 ku ngulu w’ekituli ekizibu (Ekifaananyi 3, F ne G). Ku ngulu kwa cytoplasm, Gln34 eriraanye ekibinja kya keto ekya αβ-14 carotenoids. Okugatta ku ekyo, molekyu ya n-dodecyl β-d-maltoside (β-DDM) yagonjoolwa, era omukira gwayo ogw’amazzi ne gugaziwa okutuuka ku nsengekera wakati wa puloteyina-W ne αβ-14, era omukira gw’amasavu guyinza okuba nga gusangibwa mu mubiri. Era twakiraba nti ebitundu bya C-terminal resolution ebya protein W ne RCH biri kumpi nnyo, naye si mu buwanvu bw’okukola enkolagana ezenjawulo (Figure 1, A and E). Naye wayinza okubaawo enkolagana mu amino asidi za C-terminal ezitagonjoddwa eza puloteyina zino ebbiri, eziyinza okuwa enkola y’okuwandiika puloteyina-W mu kiseera ky’okukuŋŋaanya ekizibu kya RC-LH114-W.
(A) Protein-W, etunudde mu nsengekera ne LH1αβ14 mu ngeri ya katuni, erina olujegere lw’ebbali olufaanana ng’omuggo (olumyufu), olulagibwa mu kitundu ky’ekifaananyi ky’obusobozi bw’amasannyalaze (okungulu enzirugavu entangaavu nga ku ddaala lya kontula lya 0.13). (B) Puloteeni-W ekiikirira n’enjuba eya langi etaliimu mazzi. Ebitundu bya polar ne charged biragiddwa mu cyan, ebitundu ebiziyiza amazzi biragiddwa mu langi enjeru, ate ebitundu ebirimu amazzi amangi biragiddwa mu langi ya kijanjalo. (C ne D) Puloteeni-W ekiikirira mu katuni, okutunula kwayo kwe kumu nga mu (A) (C), era nga yeekulukuunya 180° (D). Okusinziira ku kifo mu nsengekera, ebisigadde ebyawulwa bikwata ensengekera ya langi y’omusota gw’enkuba, nga N-terminal ya bbululu ate C-terminal ya langi emmyufu. (E) Puloteeni-W mu ndowooza y’emu nga mu (A), n’ebisigadde ku nsengekera ya puloteyina-W:LH1 bikiikirira emiggo egirina obubonero obugattibwako. (F) Protein-W ekyusibwakyusibwa 90° okusinziira ku (E) ne LH1αβ14 mu kifaananyi kya katuni, era okusinziira ku bisigalira by’ensengekera mu kifaananyi ky’ebbaala. Ebisigadde ebiwaniridde waggulu okuva mu beta polypeptide biwandiikibwako. Cofactor eragiddwa nga ebbaala ekwatagana ne langi y’ekifaananyi 1, β-DDM evunze eragiddwa mu langi enzirugavu, ate oxygen eragiddwa mu langi emmyufu. (G) Okulaba mu (F) kuzimbiddwa 180°, nga waliwo ebisigadde ebimanyiddwa ennyo ebya alpha polypeptide ewandiikiddwa.
Protein-W edda mu kifo kya αβ heterodimer (eya 15 mu kifaananyi 1F), bwe kityo n’eziyiza okuggalawo loopu n’okulengejja αβ heterodimers essatu ezisooka. Kyazuulibwa nti enkoona esinga obunene ey’okuserengeta eya αβ-1 heterodimer esooka okusinziira ku firimu eya bulijjo yali 25° okutuuka ku 29° (Ekifaananyi 1, A ne E), ekyakolebwa okuserengeta kwa 2° okutuuka ku 8° okwa αβ-1 mu RC A sharp contrast-LH116 (Ekifaananyi 1, B ne F). Heterodimers eyookubiri n’eyookusatu ziserengese ku 12° okutuuka ku 22° ne 5° okutuuka ku 10°. Olw’okuziyiza kwa steric okwa RC, okulengejja kwa αβ-1 tekuliimu pair ya αβ eyookubiri (ekwatagana ne αβ ey’e 16 mu kifaananyi 1F), bwe kityo ne kikola ekituli ekitegeerekeka mu mpeta ya LH1 (Ekifaananyi 1, A ne E) . Olw’obutaba na αβ heterodimers bbiri, nga ziwerekerwako okufiirwa BChl nnya ne carotenoids bbiri, tewali n’emu ku carotenoids esiba ku twisted αβ-1 subunit, ekivaamu empeta ya LH114-W erimu carotenoids 13 Vegetarian ne 28 BChls. Okubalirira kw’okusalawo kw’ekitundu okw’ebizibu ebibiri mu bitundu bya αβ1 okutuuka ku 7 kuli wansi okusinga okw’ebitundu ebirala eby’olukoba lwa LH1, ekiyinza okulaga obuveera obuzaaliranwa obw’ekitundu kya LH1 ekiriraanye ekifo kya RC QB (Ekifaananyi 4) .
Ebifaananyi bya RC-LH114-W (A ne B) ne RC-LH116 (C ne D) biragiddwa okuva mu ndabika y’emu waggulu/okulaba ku ludda (A ne B) (A ne C) n’okungulu kw’ekituli mu kifaananyi 1. (B ne D). Ebisumuluzo ebya langi biragiddwa ku ddyo.
Ekizibu ekirala kyokka eky’engeri y’omusingi ekirina omugerageranyo gwa stoichiometric ogwa 1:14 ye Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Naye, puloteyina W ne PufX tezirina nkolagana eyeyoleka, era zirina kinene kye zikola ku nsengekera zazo eza LH1. PufX ye TMH emu erimu N-terminal cytoplasmic domain ekwatagana n’oludda lwa cytoplasmic olwa RC-H subunit (13) mu kifo ekikwatagana ne Rps. ekiwuka ekiyitibwa palustris LH116αβ-16. PufX ekola omukutu gw’okuwanyisiganya quinone/quinolone wakati wa RC-LH1 ne cytochrome bcl complex era guli mu Rba zonna. sphaeroides ekizibu ky’omusingi (13). Wadde nga ensengekera ya monomera-monomer eri mu Rba. Sphaeroides RC-LH1-PufX dimer esangibwa mu kifo eky’okusiba protein W mu RC-LH114-W, era ekituli ekireetebwa PufX ne protein-W kiri mu kifo ekyenkanankana (Figure S7A). Ekituli mu RC-LH114-W nakyo kikwatagana n’omukutu gwa quinone oguteeberezebwa (8) ogwa Pseudomonas rosea LH1, ogukolebwa peptides ezitakwatagana na protein W oba PufX (Figure S7B). Okugatta ku ekyo, omukutu gwa quinone mu Blc. LH1 eya kiragala emerald ekoleddwa nga tobaliddemu ekitundu kimu ekya γ (7) esangibwa mu kifo ekifaanagana (Figure S7C). Newankubadde nga zitabaganyizibwa puloteyina ez’enjawulo, okulabika kw’emikutu gino egya quinone/quinolol mu kifo eky’awamu mu RC-LH1 complex kirabika nga kyakulabirako kya nkulaakulana ekwatagana, ekiraga nti ekituli ekitondebwawo puloteyina W kiyinza okukola ng’omukutu gwa quinone.
Ekituli mu loopu ya LH114-W kisobozesa okutondebwawo kw’ekitundu ky’olususu ekigenda mu maaso wakati w’ekifo eky’omunda eky’ekizibu kya RC-LH114-W n’oluwuzi olunene (Ekifaananyi 1G), okusinga okuyunga ebitundu bino ebibiri okuyita mu butuli bwa puloteyina nga mu puloteyina. Ekizibu kya RC-LH116 kifaananako ne Tch enzigale. Ekizibu ekiringa empiso (22) (Ekifaananyi 1H). Okuva okusaasaana kwa quinone okuyita mu luwuzi bwe kusinga okusaasaana okuyita mu mukutu gwa puloteyina omufunda, olukoba lwa LH114-W oluggule lusobola okusobozesa okukyusakyusa kwa RC okw’amangu okusinga olukoba lwa LH116 oluggaddwa, era okusaasaana kwa quinone mu RC kuyinza okuba nga kuziyiziddwa nnyo. Okusobola okugezesa oba puloteyina W ekosa okukyusa quinones okuyita mu RC, twakola cytochrome oxidation assay ku kigero ekimu ekya ubiquinone 2 (UQ2) (analogue ya natural UQ10 n’omukira gwa isoprene omumpi) (Figure 2E). Newankubadde okubeerawo kwa chelated quinone kulemesa okusalawo okutuufu okwa Michaelis constant okulabika (RC-LH114-W ne RC-LH116 zisaanira 0.2±0.1μM ne 0.5±0.2μM, respectively), omuwendo ogusinga obunene ogwa RC-LH114-W ( 4.6±0.2 e-RC-1 s-1) gwe 28±5% obunene okusinga RC-LH116 (3.6±0.1 e-RC-1 s-1).
Twasooka kuteebereza nti puloteyina-W eriwo mu bitundu nga 10% eby’ekizibu ky’omusingi ( 16 ); wano, emiwendo gy’obutoffaali obukula obw’ekitangaala ekitono, eky’ekitangaala eky’omu makkati, n’eky’ekitangaala ekinene giri 15±0.6%, 11±1% ne 0.9±0.5, okusinziira ku % (Ekifaananyi 2F). Okugerageranya omuwendo gwa mass spectrometry kwalaga nti okugattako histidine tag tekyakendeeza ku bungi bwa protein-W obw’enjawulo bw’ogeraageranya n’ebika by’omu nsiko (P = 0.59), kale emitendera gino si artifact ya protein-W ekyusiddwa (Figure S10). Naye, okutuula kuno okutono okwa puloteyina-W mu kizibu kya RC-LH1 kuyinza okusobozesa RC ezimu okukyusakyusa ku sipiidi ey’amangu, bwe kityo ne kikendeeza ku kuwanyisiganya kwa quinone/quinolone okugenda empola mu kizibu kya RC-LH116. Twakiraba nti omuwendo gw’okubeera kw’ekitangaala ekinene tegukwatagana na data ya transcriptomics eyaakafuluma, ekiraga nti pufW gene expression yeeyongera wansi w’ekitangaala eky’amaanyi (Figure S11) (23). Enjawulo wakati w’okuwandiika kwa pufW n’okuyingizibwa kwa puloteyina-W mu kizibu kya RC-LH1 etabula era eyinza okulaga okulungamya okuzibu okwa puloteyina.
Mu RC-LH114-W, molekyu za cardiolipin (CDL) 6, phosphatidylcholine (POPC) 7, phosphatidylglycerol (POPG) 1 ne β-DDM 29 zigabibwa era ne zikozesebwa mu kyokulabirako mu yo CDL 6, POPC 24, POPG 2 ne βDDM 12. RC-LH116 (Ekifaananyi 5, A ne B). Mu nsengekera zino ebbiri, CDL kumpi esangibwa ku ludda lwa cytoplasmic olw’ekizibu, ate POPC, POPG ne β-DDM zisinga kubeera ku ludda lwa luminal. Molekyulu bbiri ez’amasavu n’ez’okunaaba zaawulwa mu kitundu kya αβ-1 okutuuka ku αβ-6 eky’ekizibu kya RC-LH114-W (Ekifaananyi 5A), ate ttaano zaawulwa mu kitundu ekyenkanankana ekya RC-LH116 (Ekifaananyi 5B ). Lipids ezisingawo zasangibwa ku ludda olulala olw’ekizibu, okusinga CDL, ezikung’aanyiziddwa wakati wa RC ne αβ-7 okutuuka ku αβ-13 (Ekifaananyi 5, A ne B). Lipids endala ezigonjoddwa mu nsengekera n’eby’okunaaba zisangibwa ebweru w’empeta ya LH1, era enjegere za acyl ezigonjoddwa obulungi zigaziwa wakati wa LH1 subunits, mu kugezesebwa zimanyiddwa nga β-DDM mu RC-LH114-W, era zitegeezebwa nga β-DDM mu RC A omutabula gwa β-DDM ne POPC-LH116. Ebifo ebifaanagana eby’amasavu aga chelating n’eby’okunaaba mu nsengekera yaffe biraga nti bifo bikwatagana mu mubiri (Figure S12A). Ebifo bya molekyo ezenkanankana mu Tch nabyo birina okukwatagana okulungi. Gentle ne Trv. Ekika 970 RC-LH1s (Ekifaananyi S12, B okutuuka ku E) (9, 12) n’ebisigadde ebikwatagana ne haidrojeni eby’ekibinja ky’omutwe gw’amasavu byalaga okukuuma okulungi ennyo mu kukwatagana kw’omutendera (Ekifaananyi S13), ekiraga nti Conserved CDL esiba ku RC (24), ebifo bino biyinza okukuumibwa mu kizibu kya RC-LH1.
(A ne B) RC-LH114-W (A) ne RC-LH116 (B) peptides zikiikirira katuni, ate langi zikiikirira emiggo, nga tukozesa ensengeka ya langi mu kifaananyi 1. Amasavu galagibwa mu langi emmyufu, ate eby’okunaaba biragiddwa mu langi enzirugavu. UQ esibiddwa ku bifo bya RC QA ne QB ya kyenvu, ate UQ eyawuddwamu ya bbululu. (C ne D) Endowooza ze zimu nga (A) ne (B), nga lipids zirekeddwawo. (E okutuuka ku G) Okulaba okugaziyiziddwa kwa Q1(E), Q2(F) ne Q3(G) okuva ku RC-LH116, nga kuliko enjegere ez’ebbali ezikwata ku ndala. Ensigo za haidrojeni ziragiddwa nga layini enjeru eziriko enkokola.
Mu RC-LH116, RC QA ne QB UQ zombi, ezeetabye mu kutambuza obusannyalazo mu nkola y’okwawula ekisannyalazo, zivunda mu bifo byabwe eby’okusiba. Wabula mu RC-LH114-W, QB quinone tennagonjoolwa era ejja kwogerwako mu bujjuvu wansi. Ng’oggyeeko QA ne QB quinones, molekyo za UQ bbiri eziriko chelated (ezisangibwa wakati w’empeta za RC ne LH1) zigabanyizibwa mu nsengekera ya RC-LH114-W okusinziira ku bibinja by’emitwe gyazo ebigonjoddwa obulungi (ezisangibwa mu Q1 ne Q2, mu kulondako). ekifo). Ekifaananyi 5C). Yuniti za isoprene bbiri ziweebwa Q1, era maapu ya density egonjoola emikira gya isoprene 10 emijjuvu egya Q2. Mu nsengekera ya RC-LH116, molekyo za UQ10 ssatu eza chelated (Q1 okutuuka ku Q3, Ekifaananyi 5D) zagonjoolwa, era molekyo zonna zirina density entegeerekeka mu mukira gwonna (Figure 5, D to G). Mu nsengekera zombi, ebifo by’ebibinja by’emitwe gya quinone ebya Q1 ne Q2 birina okukwatagana okulungi ennyo (Ekifaananyi S12F), era bikwatagana ne RC yokka. Q1 esangibwa ku mulyango gw’ekituli kya W ekya RC-LH114-W (Ekifaananyi 1G ne 5, C, D ne E), ate Q2 esangibwa okumpi n’ekifo QB we basiba (Ekifaananyi 5, C, D) ne F). Ebisigadde bya L-Trp143 ne L-Trp269 ebikuumiddwa biri kumpi nnyo ne Q1 ne Q2 era biwa enkolagana eziyinza okubaawo π-stacking (Ekifaananyi 5, E ne F, n’Ekifaananyi S12). L-Gln88, 3.0 Å okuva ku okisigyeni ow’ewala eya Q1, egaba ekiyungo kya haidrojeni eky’amaanyi (Ekifaananyi 5E); ekisigadde kino kikuumibwa mu RC zonna okuggyako enkolagana esinga okuba ewala (Ekifaananyi S13). L-Ser91 ekyusibwakyusibwa mu ngeri ey’okukuuma Thr mu RC endala ezisinga obungi (Ekifaananyi S13), eri 3.8 Angstroms okuva mu methyl oxygen ya Q1, era eyinza okuwa enkolagana za haidrojeni enafu (Ekifaananyi 5E). Q3 telabika ng’erina nkolagana eyenjawulo, wabula esangibwa mu kitundu ekiziyiza amazzi wakati wa RC-M subunit ne LH1-α subunit 5 okutuuka ku 6 (Ekifaananyi 5, D ne G). Q1, Q2 ne Q3 oba quinones eziriraanye chelated nazo zigonjoddwa mu Tch. Omugonvu, Trv. Ekika 970 ne Blc. Ensengekera ya iris (9, 10, 12) eraga ekifo ekikuumibwa eky’okusiba quinone omuyambi mu RC-LH1 complex (Figure S12G). UQs ettaano ezivunze mu RC-LH116 zikwatagana bulungi ne 5.8±0.7 eza buli complex ezisaliddwawo high performance liquid chromatography (HPLC), ate UQs essatu ezivunze mu RC-LH114-W ziri wansi okusinga Omuwendo ogupimiddwa ogwa 6.2±0.3 (Fig. S14) gulaga nti waliwo molekyu za UQ ezitagonjoddwa mu nsengekera.
Pseudo-symmetric L ne M polypeptides buli emu erimu TMH ttaano era zikola heterodimer egatta BChl dimer emu, BChl monomers bbiri, bacteriophage (BPh) monomers bbiri, n’ekyuma kimu ekitali kya Heme ne molekyu emu oba bbiri eza UQ10. Okuyita mu kubeerawo kw’enkolagana ya haidrojeni ku kibinja kya ketoni ekisembayo n’okukuŋŋaanyizibwa kwayo okumanyiddwa mu Rps, karotenoyidi ziyingizibwa mu M-subunit, eyitibwa cis-3,4-dehydroorhodopin. Ebika (25). Ekitundu ky’oluwuzi olw’ebweru ekya RC-H kinywezebwa ku luwuzi nga TMH emu. Ensengekera ya RC okutwalira awamu efaananako ne subunit essatu RC ez’ebika ebikwatagana (nga Rba). ebiwuka ebiyitibwa sphaeroides (PDB ID: 3I4D). Macrocycles za BChl ne BPh, carotenoid backbone ne non-heme iron zikwatagana mu resolution range y’ensengekera zino, nga bwe kiri mu kibinja ky’omutwe gwa UQ10 mu kifo kya QA ne QB quinone ku RC-LH116 (Figure S15).
Okubeerawo kw’ebizimbe bibiri ebya RC ebirina emiwendo egy’enjawulo egy’okubeera mu kifo kya QB kiwa omukisa omupya okwekenneenya enkyukakyuka z’ensengekera ezitakyukakyuka eziwerekera okusiba kwa QB quinone. Mu kizibu kya RC-LH116, QB quinone esangibwa mu kifo “eky’okumpi” ekisibiddwa mu bujjuvu (26), naye okwawukana kwa RC-LH114-W tekulina QB quinone. Tewali QB quinone mu RC-LH114-W, ekyewuunyisa kubanga ekizibu kikola, okusinga ekizibu kya RC-LH116 ekirimu QB quinone egonjoddwa mu nsengeka. Newankubadde empeta za LH1 ebbiri zikola chelate nga quinones mukaaga, ttaano zigonjoolwa mu nsengekera mu mpeta ya RC-LH116 enzigale, ate ssatu zokka ze zikoma mu nsengeka mu mpeta ya RC-LH114-W enzigule . Obuzibu buno obw’enzimba obweyongedde buyinza okulaga okukyusa amangu ebifo bya RC-LH114-W QB, kinetics ya quinone ey’amangu mu complex, n’okwongera ku mikisa gy’okusala loopu ya LH1. Tuteesa nti obutaba na UQ mu kifo kya RC QB ekya RC-LH114-W kiyinza okuba nga kiva ku kizibu ekizibu ennyo era ekikola ennyo, era ekifo kya QB ekya RC-LH114-W kibadde kifuuse bbugumu amangu ddala mu nkyukakyuka ya UQ Omutendera ogw’enjawulo (omulyango oguyingira mu kifo kya QB guggaddwa) gulaga ensengekera y’omulimu guno.
Awatali QB, okukyukakyuka okuwerekera kwa L-Phe217 okutuuka mu kifo ekitakwatagana na kusiba kwa UQ10, kubanga kujja kuleeta okutomeragana okw’ekifo ne yuniti ya isoprene esooka ey’omukira (Ekifaananyi 6A). Okugatta ku ekyo, enkyukakyuka enkulu ezeeyolese mu nsengekera zeeyoleka, naddala helix de (helix ennyimpi mu loopu wakati wa TMH D ne E) nga L-Phe217 ekyusibwa okudda mu nsawo esiba QB n’okuzimbulukuka kwa L-Tyr223 (Ekifaananyi 6A ) Okumenya ekiyungo kya haidrojeni n’ensengekera ya M-Asp45 n’okuggala omulyango gw’ekifo ekisiba QB (Ekifaananyi 6B). Helix de pivots ku base yaayo, Cα ya L-Ser209 ekyusibwa 0.33Å, ate L-Val221Cα ekyusibwa 3.52Å. Tewali nkyukakyuka zirabika mu TMH D ne E, eziteekebwa waggulu mu nsengekera zombi (Ekifaananyi 6A). Okusinziira ku kye tumanyi, eno ye nsengeka esooka mu RC ey’obutonde eggalawo ekifo kya QB. Okugerageranya n’ensengekera enzijuvu (QB-bound) kulaga nti nga quinone tennakendeezebwa, enkyukakyuka mu nsengekera yeetaagibwa okugifuula okuyingira mu quinone. L-Phe217 yeekulukuunya okukola enkolagana ya π-stacking n’ekibinja ky’omutwe gwa quinone, era helix ekyuka okudda ebweru, ekisobozesa amagumba ga L-Gly222 n’olujegere olw’ebbali olwa L-Tyr223 okukola omukutu gw’enkolagana ya haidrojeni nga gulina ensengekera y’enkolagana ya haidrojeni ennywevu (Ekifaananyi 6, A ne C).
(A) Katuuni ekwatagana eya hologram (L chain, orange/M chain, magenta) ne apo (gray) structure, nga mu kino ebisigadde ebikulu biragibwa mu ngeri y’ekifaananyi ekiringa omuggo. UQ10 ekiikirira ebbaala eya kyenvu. Layini eriko ennukuta eraga enkolagana za haidrojeni ezikoleddwa mu nsengekera yonna. (B ne C) Ekifaananyi eky’okungulu ekya apolipoprotein n’ensengekera y’empeta yonna, nga kiraga okisigyeni w’olujegere olw’ebbali olwa L-Phe217 mu bbululu ne L-Tyr223 mu langi emmyufu, mu kulondako. Ekitundu kya L kya langi ya kijanjalo; ebitundu ebitono ebya M ne H tebirina langi. .
Mu ngeri etasuubirwa, newankubadde nga ensengekera eziwerako eza RC ezibulamu QB ezitaliiko LH1 ziriwo, enkyukakyuka mu nsengekera (conformational changes) ezirabibwa mu kunoonyereza kuno tezinnaba kuloopebwa. Mu bino mulimu ensengeka y’okukendeeza QB okuva mu Blc. viridis (ID ya PDB: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) ne Rba. sphaeroides (PDB ID: 1OGV) (29), byonna kumpi bye bimu n’ensengekera yaabwe eya QB okutwalira awamu. Okukebera obulungi 3PRC kwazuula nti molekyu z’eky’okunaaba eza LDAO (Lauryl Dimethyl Amine Oxide) zisiba ku mulyango gw’ekifo kya QB, ekiyinza okulemesa okuddamu okusengeka mu nsengekera enzigale. Wadde nga LDAO tevunda mu kifo kye kimu mu 1EYS oba 1OGV, RC zino zitegekebwa nga tukozesa eky’okunaaba kye kimu n’olwekyo ziyinza okuvaamu ekikolwa kye kimu. Ensengekera ya kirisitaalo ya Rba. Sphaeroides RC co-crystallized ne cytochrome c2 (PDB ID: 1L9B) nayo erabika ng’erina ekifo kya QB ekiggaddwa. Naye, mu mbeera eno, ekitundu kya N-terminal ekya RC-M polypeptide (ekikwatagana n’ekifo ekisiba QB okuyita mu kiyungo kya H eky’ekisigadde kya Tyr ku Q helix) kikwata ensengekera etali ya butonde, era enkyukakyuka y’ensengekera ya QB teyongera kunoonyerezebwako (30 ). Ekigumya kiri nti tetulabye kika kino eky’okukyukakyuka kwa M polypeptide mu nsengekera ya RC-LH114-W, kumpi kye kimu n’ekitundu kya N-terminal ekya RC-LH116 RC. Era kisaana okumanyibwa nti oluvannyuma lw’okumalawo antenna ya LH1 eyesigamiziddwa ku detergent, apolipoprotein RCs mu PDB zagonjoolwa, ekyamalawo ebidiba bya quinone eby’omunda n’amasavu mu bbanga wakati wa RC n’oludda olw’omunda olw’empeta ya LH1 eyeetoolodde ( 31 , 32 ). RC esigala nga ekola kubanga ekuuma cofactors zonna, okuggyako QB quinone evunda, etali nnywevu nnyo era etera okubula mu nkola y’okuteekateeka ( 33 ). Okugatta ku ekyo, kimanyiddwa nti okuggyawo LH1 n’amasavu ag’obutonde ag’enzirukanya okuva mu RC kuyinza okuba n’akakwate ku mirimu, gamba ng’obulamu obutono obw’embeera ya P+QB eyawuddwamu omusannyalazo ( 31 , 34 , 35 ). N’olwekyo, tuteebereza nti okubeerawo kw’empeta ya LH1 ey’ekitundu eyeetoolodde RC kuyinza okukuuma ekifo kya QB “ekiggaddwa”, bwe kityo ne kikuuma embeera y’ekitundu okumpi ne QB.
Newankubadde nga apolipoprotein (etaliimu QB quinone) n’ensengekera enzijuvu bikiikirira ebifaananyi bibiri byokka eby’enkyukakyuka y’ekifo kya QB, okusinga ebibaddewo ebiddiriŋŋana, waliwo ebiraga nti okusiba kuyinza okuteekebwako enzigi okuziyiza okuddamu okusiba nga hydroquinone Okuziyiza okuziyiza kwa substrate. Enkolagana ya quinolol ne quinone okumpi n’ekifo kya QB ekya apolipoprotein eyinza okuba ey’enjawulo, ekivaako okugaanibwa kwayo RC. Kibadde kiteesebwa okuva edda nti enkyukakyuka mu nsengekera (conformational changes) zikola kinene mu kusiba n’okukendeeza ku quinones. Obusobozi bwa RCs ezifumbiddwa okukendeeza ku quinones oluvannyuma lw’okutuukagana n’ekizikiza bukosebwa (36); X-ray crystallography eraga nti okwonooneka kuno kuva ku QB quinones okusibira mu “distal” conformation nga 4.5 Å okuva active proximal position (26) , 37). Tuteesa nti ensengekera eno ey’okusiba ewala kifaananyi kya mbeera ey’omu makkati wakati wa apolipoprotein n’ensengekera y’empeta enzijuvu, ekigoberera enkolagana esooka ne quinone n’okugguka kw’ekifo kya QB.
Ekika kya II RC ekisangibwa mu PSII complex ya bakitiriya ezimu ezikola ekitangaala ne cyanobacteria, algae n’ebimera kirina okukuuma enzimba n’emirimu ( 38 ). Ensengeka y’enzimba eragiddwa mu kifaananyi 6 (D ne E) eggumiza okufaanagana wakati wa PSII RCs n’ekifo kya QB eky’ekizibu kya RC ekya bakitiriya. Okugerageranya kuno kumaze ebbanga nga kubadde kyakulabirako mu kusoma enkola ezikwatagana ennyo ez’okusiba n’okukendeeza kwa quinone. Ebiwandiiko eby’emabega byalaga nti enkyukakyuka mu nsengekera (conformational changes) ziwerekerwako okukendeeza kwa PSII kwa quinones ( 39 , 40 ). N’olwekyo, okusinziira ku kukuuma RC mu nkulaakulana, enkola eno ey’okusiba eyali tetulabiddwako eyinza n’okukozesebwa ku kifo kya QB ekya PSII RC mu bimera ebikola ekitangaala ebirimu omukka.
Rps ΔpufW (okuggyibwako pufW okutaliiko kabonero) ne PufW-His (C-terminal 10x His-tagged protein-W eraga okuva mu kifo kya pufW eky’obutonde) ebika. palustris CGA009 yayogerwako mu mulimu gwaffe ogwasooka ( 16 ). Ebika bino n’omuzadde ow’ekika ky’omu nsiko ow’ekika kya isogenic byaggyibwa mu firiiza nga bakuba obutoffaali obutono ku PYE (buli emu liita 5 g -1) (obuterekeddwa mu LB ku -80 °C, nga mulimu 50% ( w/v) glycerol) protein, yeast extract ne succinate) agar [1.5% (w/v)] plate. Ebbakuli yafumbirwa ekiro kyonna mu nzikiza ku bbugumu erya bulijjo mu mbeera ezitaliimu mukka, n’oluvannyuma n’eyaka n’ekitangaala ekyeru (~50 μmolm-2 s-1) ekyaweebwa bbaatule za OSRAM 116-W halogen bulbs (RS Components, UK) okumala ennaku 3 ku 5 okutuusa nga ekibinja kimu kyalabika. Koloni emu yakozesebwa okugema 10 ml za M22+ medium (41) nga zigattako 0.1% (w/v) casamino acids (wano ziyitibwa M22). Obuwangwa bwakuzibwa mu mbeera ya oxygen omutono mu nzikiza ku 34°C nga bukankanyizibwa ku 180 rpm okumala essaawa 48, n’oluvannyuma 70 ml z’obuwangwa ne zisiigibwa mu mbeera y’emu okumala essaawa 24. Ekika kya semi-aerobic culture nga kirimu obuzito bwa 1 ml kikozesebwa okugema 30 ml za M22 medium mu 30 ml universal screw-top transparent glass bottle era ne kiweebwa obusannyalazo n’okutabula (~50μmolm-2 s-1) okumala essaawa 48 nga bakozesa amaanyi ga magineeti agataliimu Stirring rod. Olwo 30 ml z’obuwangwa zaasiimibwa ne liita nga 1 ez’obuwangwa mu mbeera ze zimu, oluvannyuma ne zikozesebwa okusiiga liita nga 9 ez’obuwangwa nga zitangalijja ku ~200 μmolm-2 s-1 okumala essaawa 72. Obutoffaali bwakungula nga buyita mu kusengejja ku 7132 RCF okumala eddakiika 30, ne buddamu okusibwa mu ~10 ml za 20 mM tris-HCl (pH 8.0), ne buterekebwa ku -20°C okutuusa lwe kyetaagisa.
Oluvannyuma lw’okusaanuuka, ssaako obuwundo obumu obwa deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) n’empeke bbiri eza Roche holoenzyme protease inhibitor (Merck, UK) mu butoffaali obuzzeemu okusibwa. Mu 20,000 psi French pressure cell (Aminco, USA), obutoffaali bwataataaganyizibwa emirundi 8 ku 12. Oluvannyuma lw’okuggyawo obutoffaali obutamenyese n’ebisasiro ebitasaanuuka nga tuyita mu kusengejja ku 18,500 RCF okumala eddakiika 15 ku 4°C, oluwuzi lwatonnya okuva mu lysate eya langi nga luyita mu kusengejja ku 113,000 RCF okumala essaawa 2 ku 43,000°C. Suula ekitundu ekisaanuuka era oddemu okuwanirira oluwuzi olwa langi mu 100 ku 200 ml za 20 mM tris-HCl (pH 8.0) era oziteeke emu okutuusa nga tewali bikozesebwa birabika. Olususu oluyimiriziddwa lwafumbirwa mu 20 mM tris-HCl (pH 8.0) (Anatrace, USA) nga mulimu 2% (w/v) β-DDM okumala essaawa 1 mu nzikiza ku 4°C nga tutabuddwa mpola. Oluvannyuma centrifuge ku 70°C okusaanuusa 150,000 RCF ku 4°C okumala essaawa 1 okuggyawo ebisigadde ebitasaanuuka.
Olususu olusaanuuka okuva mu kika kya ΔpufW lwasiigibwa ku 50 ml DEAE Sepharose ion exchange column nga erimu volumes ssatu ez’ennyiriri (CV) eza binding buffer [20 mM tris-HCl (pH 8.0) ezirimu 0.03% (w / v) β-DDM]. Naaba empagi n’ebiziyiza bibiri ebisiba CV, n’oluvannyuma onaabe empagi n’ebiziyiza bibiri ebisiba ebirimu 50 mM NaCl. Ekizibu kya RC-LH116 kyasengejebwa n’ekigerageranyo kya linear gradient ekya 150 okutuuka ku 300 mM NaCl (mu binding buffer) ku 1.75 CV, ate ekizibu ky’okusiba ekyasigalawo kyasengejebwa ne binding buffer erimu 300 mM NaCl ku 0.5 CV. Kuŋŋaanya ekisengejjo ky’okunyiga wakati wa 250 ne 1000 nm, kuuma ekitundu ekirina omugerageranyo gw’okunyiga (A880/A280) ogusinga 1 ku 880 ku 280 nm, kifukirire emirundi ebiri mu buffer y’okusiba, era oddemu okozese enkola y’emu ku mpagi ya DEAE On purification. Fuumuula obutundutundu obulina emigerageranyo gya A880/A280 egya waggulu okusinga 1.7 ne A880/A805 egya waggulu okusinga 3.0, kola lawundi eyookusatu ey’okuwanyisiganya ion, era osigame obutundutundu obulina emigerageranyo gya A880/A280 egya waggulu okusinga 2.2 ne A880/A805 emigerageranyo waggulu okusinga 5.0. Ekizibu ekirongooseddwa ekitundu kyasengejebwa okutuuka ku ~2 ml mu Amicon 100,000 molecular weight cut-off (MWCO) centrifugal filter (Merck, UK), ne kitikkibwa ku Superdex 200 16/600 size exclusion column (GE Healthcare, US) erimu 200 mM NaCl buffer, n’oluvannyuma ne kifukibwa mu buffer y’emu ku 1.5 CV. Kuŋŋaanya spektira z’okunyiga ez’ekitundu ekiggyako obunene, era osengeke spektira z’okunyiga n’emigerageranyo gya A880/A280 ku 2.4 ne A880/A805 emigerageranyo ku 5.8 okutuuka ku 100 A880, era amangu ago zikozese okuteekateeka oba okutereka giridi ya cryo-TEM Kuuma ku -80°C okutuusa nga kyetaagisa.
Olususu olusaanuuka okuva mu kika kya PufW-His lwasiigibwa ku 20 ml HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) erimu 200 mM NaCl ne 0.03% (w/w)) mu IMAC buffer ( GE Healthcare). v) β-DDM] nga bwe kiri. Empagi yanaazibwa ne CV ttaano eza IMAC buffer, n’oluvannyuma ne CV ttaano eza IMAC buffer ezirimu 10 mM histidine. Ekizibu ky’omutwe (core complex) kyasengejebwa okuva mu mpagi ne IMAC buffers ttaano ezirimu 100 mM histidine. Ekitundu ekirimu RC-LH114-W complex kisengejebwa okutuuka ku ~10 ml mu ttanka etabuddwamu erimu Amicon 100,000 MWCO filter (Merck, UK), ne kifukibwa emirundi 20 ne binding buffer, n’oluvannyuma ne kigattibwa ku 25 ml Mu DEAE Sepharose column, CV nnya ezisibiddwa ku buffer zikozesebwa nga bukyali. Naaba empagi ne buffers nnya ezisiba CV, olwo olongoose ekizibu ku CV munaana ku linear gradient ya 0 okutuuka ku 100 mM NaCl (mu buffer esiba), ne CVs ennya ezisigadde nga zirimu 100 mM binding buffer Ebizibu ebisigadde ebisengejeddwa ku sodium chloride nga bigattiddwa n’omugerageranyo gwa A880/A280 ogusinga 2.4 n’o Omugerageranyo gwa A880/A805 ogusinga obutundutundu 4.6 gwasengejebwa okutuuka ku ~2 ml mu Amicon 100,000 MWCO centrifugal filter, ne gujjula 1.5 CV IMAC nga bukyali Buffer equilibrated Superdex 200 16/600 size exclusion column, n’oluvannyuma ne gusengejebwa mu buffer y’emu ku 1.5 CV. Kuŋŋaanya spektira z’okunyiga ez’obutundutundu obuggyibwako obunene n’okugatta spektira z’okunyiga n’emigerageranyo gya A880/A280 ku 2.1 ne A880/A805 emigerageranyo ku 4.6 okutuuka ku 100 A880, ezikozesebwa amangu ddala okuteekateeka ekisenge kya TEM ekifumbiddwa oba okuterekebwa ku -80°C okutuusa lwe weetaaga.
Freezer ya Leica EM GP immersion freezer yakozesebwa okuteekateeka TEM grids ez’ebbugumu eri wansi. Ekizibu kino kyafukibwa mu IMAC buffer okutuuka ku A880 ya 50, n’oluvannyuma 5μl ne zitikkibwa ku katimba k’ekikomo akaakafuluma akatangalijja aka QUANTIFOIL 1.2/1.3 akasiigiddwa kaboni (Agar Scientific, UK). Fumbira giridi ku 20°C ne 60% relative humidity okumala s 30, olwo ogisiige okukala okumala s 3, n’oluvannyuma ogizikize mu liquid ethane ku -176°C.
Data y’ekizibu kya RC-LH114-W yawandiikibwa ku eBIC (Electronic Bioimaging Center) (British Diamond Light Source) n’ekintu ekirabika obulungi ekya Titan Krios, ekikola ku vvulovumenti ey’amangu eya 300kV, ng’erina okukuza okw’erinnya okwa 130,000× n’amasoboza ga- Londa ekituli kya 20 eV. Gatan 968 GIF Quantum nga erina K2 peak detector yakozesebwa okuwandiika ebifaananyi mu mode y’okubala okukung’aanya data. Sayizi ya pikseli ekaliddwa eri 1.048Å, ate omuwendo gwa ddoozi guli 3.83 e-Å-2s-1. Firimu yagikung’aanya mu sikonda 11 n’agigabanyaamu ebitundu 40. Kozesa ekitundu ekisiigiddwa kaboni okuddamu okutunuulira microscope, n’oluvannyuma okuŋŋaanya firimu ssatu buli kinnya. Okutwalira awamu, firimu 3130 ze zaakuŋŋaanyizibwa, nga emiwendo gya defocus giri wakati wa -1 ne -3μm.
Ebikwata ku RC-LH116 complex byakuŋŋaanyizibwa nga tukozesa microscope y’emu mu Asterbury Biostructure Laboratory (University of Leeds, UK). Ebiwandiiko byakuŋŋaanyizibwa mu ngeri y’okubala nga bikuziddwa 130 k, era obunene bwa pikseli bwapimibwa okutuuka ku 1.065 Å nga ddoozi ya 4.6 e-Å-2s-1. Firimu eno yakwatibwa mu sikonda 12 era n’egabanyizibwamu ebitundu 48. Okutwalira awamu, firimu 3359 ze zaakuŋŋaanyizibwa, nga emiwendo gya defocus giri wakati wa -1 ne -3μm.
Okukola ku data kwonna kukolebwa mu payipu ya Relion 3.0 (42). Kozesa Motioncorr 2 (43) okutereeza entambula y’ebikondo ng’okozesa obuzito bwa ddoozi, n’oluvannyuma kozesa CTFFIND 4.1 (44) okuzuula CTF (contrast transfer function) parameter. Ebifaananyi ebitonotono ebimanyiddwa oluvannyuma lw’emitendera gino egy’okulongoosa egy’olubereberye biragiddwa mu kifaananyi 2. S16. Ekifaananyi ky’okulonda mu ngeri ey’otoma kikolebwa nga kilonda mu ngalo ppikisi nga 250 ez’obutundutundu 1000 mu fuleemu ya ppikisi 250 era nga tewali nsengeka ya reference two-dimensional (2D), bwe kityo ne kigaana ensengeka ezo ezituukana n’obucaafu bwa sampuli oba ezitaliiko mpisa zitegeerekeka. Oluvannyuma, okulonda okw’otoma kwakolebwa ku bifaananyi byonna ebitonotono, era RC-LH114-W yali ya butundutundu 849,359, ate RC-LH116 complex yali ya butundutundu 476,547. Obutoffaali bwonna obulondeddwa buyise mu lawundi bbiri ez’okugabanya 2D okutali kwa kujuliza, era oluvannyuma lwa buli kudduka, obutundutundu obusisinkana ekitundu kya kaboni, obucaafu bwa sampuli, obutabaawo bifaananyi byeyoleka oba obutundutundu obukwatagana ennyo bugaanibwa, ekivaamu 772,033 (90.9%) ne 359,678 (75.5%) ) Obutundutundu bukozesebwa mu kusengeka kwa 3D RC-LH114-W ne RC-LH116. Enkola ya 3D reference model eyasooka yakolebwa nga tukozesa enkola ya stochastic gradient descent. Nga tukozesa ekifaananyi ekisookerwako ng’ekijulizi, obutundutundu obulondeddwa bugabanyizibwa mu biti bina mu 3D. Nga okozesa model mu kiti kino nga reference, kola 3D refining ku particles mu category esinga obunene, olwo kozesa initial 15Å low-pass filter okubikka ekitundu solvent, yongera pixels 6 soft edges, era post-process pixels okutereeza Gatan K2 peak Modulation transfer function of the top detector. Ku RC-LH114-W dataset, model eno eyasooka yakyusibwa nga eggyawo density ey’amaanyi ku mbiriizi za masiki (ekutuddwa ku core complex density mu UCSF Chimera). Ebikozesebwa ebivaamu (ebisalawo bya RC-LH114-W ne RC-LH116 biri 3.91 ne 4.16 Å, mu kulondako) bikozesebwa ng’ekijuliziddwa ku lawundi eyookubiri ey’okugabanya 3D. Obutoffaali obukozesebwa bukuŋŋaanyizibwa mu kiraasi ya 3D esooka era tebulina nkolagana ya maanyi na kitundu. Okukwatagana oba obutaba na bifaananyi bya nsengeka ebyeyoleka. Oluvannyuma lw’oluzannya olwokubiri olw’okugabanya mu 3D, ekika ekisinga okuba n’obulungi bwalondebwa [Ku RC-LH114-W, ekika kimu kiri 377,703 obutundutundu (44.5%), ku RC-LH116, waliwo ebika bibiri, nga byonna awamu biweza obutundutundu 260,752 (54.7%) , Awali bwe bimu nga bikwatagana oluvannyuma lw’okuzimbulukuka okusooka nga waliwo enjawulo entono]. Obutundutundu obulondeddwa buddamu okuggyibwa mu bbokisi ya ppikisi 400 ne bulongoosebwa nga bulongoosebwa mu ngeri ya 3D. Masiki y’ekizimbulukusa ekolebwa nga tukozesa omusengejja ogusooka ogwa 15Å low-pass filter, okugaziya maapu ya pixel 3 ne masiki soft ya pixel 3. Nga tukozesa okulongoosa CTF buli kitundutundu, okutereeza entambula ya buli kitundutundu n’oluzannya olwokubiri olw’okulongoosa CTF buli kitundutundu, okulongoosa mu 3D, okusiba ekisengejja n’okulongoosa oluvannyuma lwa buli mutendera okwongera okulongoosa obutonde obuvaamu. Nga tukozesa omuwendo gw’okusalako ogwa FSC (Fourier Shell Correlation Coefficient) ogwa 0.143, okusalawo kw’ebikozesebwa ebisembayo ebya RC-LH114-W ne RC-LH116 biri 2.65 ne 2.80Å, okusinziira ku. FSC curve y’ekyokulabirako ekisembayo eragiddwa mu kifaananyi 2. S17.
Ensengekera za puloteyina zonna ziwanulibwa okuva mu UniProtKB: LH1-β (PufB; UniProt ID: Q6N9L5); LH1-α (PufA; ID ya UniProt: Q6N9L4) nga bwe kiri; RC-L (PufL; ID ya UniProt: O83005); RC-M (PufM; ID ya UniProt: A0A4Z7); RC-H (PuhA; ID ya UniProt: A0A4Z9); Puloteeni-W (PufW; ID ya UniProt: Q6N1K3). SWISS-MODEL (45) yakozesebwa okuzimba ekifaananyi kya homology model ekya RC, ekirimu ensengekera za puloteyina za RC-L, RC-M ne RC-H n’ensengekera ya kirisitaalo ya Rba. sphaeroides RC yakozesebwa nga ekifaananyi (PDB ID: 5LSE) ( 46 ). Kozesa ekintu “fit map” mu UCSF Chimera okutuukanya model ekoleddwa ku maapu (47), okulongoosa ensengekera ya puloteyina, ne cofactor [4×BChl a (monomer library residue name = BCL), 2×BPh a (BPH), ekika kimu oba bibiri ebya UQ10 (U10), ekyuma ekimu ekitali kya heme (Fe) n’ekimu 3,4-dihydrohexacarbonylcholine (QAK)] kozesa Coot (48) okugattako. Okuva QAK bweri mu tterekero lya monomer, ya parameterized nga ekozesa eLBOW tool mu PHENIX ( 49 ).
Ekiddako, ekitundu kya LH1 kyazimbibwa. Mu kusooka, ekintu ekizimba mu ngeri ey’otoma mu PHENIX (49) kyakozesebwa okuzimba ekitundu ky’omutendera gwa LH1 mu ngeri ey’otoma nga tukozesa maapu n’ensengekera za puloteyina ya LH1-α ne LH1-β ng’ebiyingizibwa. Londa subunit ya LH1 esinga okujjuvu, giggyemu ogitikkire mu Coot, yongera mu ngalo omutendera ogubula mu yo, era mu ngalo olongoose ensengekera yonna nga tonnaba kwongerako BCls bbiri a (BCL) ne spirilloxanthin (CRT) [okusinziira ku Rps ezikwatagana Densite y’ekizibu kya LH1 n’ebirimu carotenoid ebimanyiddwa. Ebika (17)]. Koppa ekitundu kya LH1 ekijjuvu, era okozese UCSF Chimera “Docking Map Tool” okusimba mu kitundu ekiriraanyewo ekitali kya model ekya LH1 density, n’oluvannyuma okirongoose mu Coot; ddiŋŋana enkola eno okutuusa nga subunits zonna eza LH1 ziwedde okukolebwako model. Ku nsengekera ya RC-LH114-W, nga tuggyamu density etagabanyizibwa mu Coot, puloteyina ekutulwamu okuva mu bitundu ebisigadde ebitali bya puloteyina mu maapu ya USCF Chimera era ekintu kya Autobuild kikozesebwa okuteekawo omuze ogusooka, n’ebitundu ebitono ebisigadde (protein-W) Modeling. Mu PHENIX (49) nga bwe kiri. Yongera ensengekera zonna ezibula ku muze oguvuddemu mu Coot (48), n’oluvannyuma okulongoosa mu ngalo ekitundu ekitono kyonna. Densite esigadde etagabanyizibwa ekwatagana n’omugatte gw’amasavu (PDB monomer library ID of CDL = CDL, POPC = 6PL ne POPG = PGT), β-DDM detergent (LMT) ne UQ10 molecules (U10). Kozesa PHENIX optimization (49) ne manual optimization mu Coot (48) okutuukiriza model esooka enzijuvu okutuusa ng’ebibalo by’ekyokulabirako n’omutindo gw’okulaba ogw’okukwatagana tebisobola kwongera kulongoosebwa. Mu kusembayo, kozesa LocScale (50) okusaza maapu y’ekitundu, n’oluvannyuma okole enzirukanya endala eziwerako ez’okukola modeling ya density etagabanyizibwa n’okulongoosa otomatiki n’omu ngalo.
Peptides, cofactors ne lipids endala ne quinones ezisimbye mu densities zazo ziragiddwa mu bifaananyi 1 ne 2. S18 okutuuka ku S23. Amawulire agakwata ku bibalo by’ekyokulabirako ekisembayo galagiddwa mu kipande S1.
Okuggyako nga kirambikiddwa bulala, ebifaananyi by’okunyiga UV/Vis/NIR byakuŋŋaanyizibwa ku Cary60 spectrophotometer (Agilent, USA) ku bbanga lya 1 nm okuva ku 250 nm okutuuka ku 1000 nm n’obudde bw’okugatta bwa 0.1s.
Fuula sampuli mu quartz cuvette ng’olina ekkubo lya mm 2 okutuuka ku A880 ya 1, era okuŋŋaanya spectrum y’okunyiga wakati wa 400 ne 1000 nm. Ensengekera za dichroic ezeekulungirivu zaakuŋŋaanyizibwa ku Jasco 810 spectropolarimeter (Jasco, Japan) ku 1 nm intervals wakati wa 400 nm ne 950 nm ku scan rate ya 20 nm min-1.
Omugerageranyo gw’okusaanawo kw’amagulu gusalibwawo nga tufuuwa ekizibu ky’omusingi okutuuka ku A880 nga 50. Fuumuula obuzito bwa 10μl mu 990μl binding buffer oba methanol, era okukuŋŋaanya spectrum y’okunyiga amangu ddala okukendeeza ku kuvunda kwa BChl. Ebirimu BChl mu buli sampuli ya methanol byabalirirwa omugerageranyo gw’okusaanawo ku 771 nm ogwa 54.8 mM-1 cm-1, era omugerageranyo gw’okusaanawo gwazuulibwa ( 51 ). Gabana ekipimo kya BChl ekipimiddwa ku 32 (RC-LH114-W) oba 36 (RC-LH116) okuzuula ekirungo kya core complex, oluvannyuma ekikozesebwa okuzuula spectrum y’okunyiga eya sampuli y’emu ekuŋŋaanyiziddwa mu buffer Extinction coefficient. tebikwatagana. Ebipimo bisatu ebiddiŋŋanwa byatwalibwa ku buli sampuli, era okunyiga okwa wakati okwa BChl Qy maximum kwakozesebwa okubala. Omugerageranyo gw’okusaanawo kwa RC-LH114-W ogupimiddwa ku 878 nm guli 3280±140 mM-1 cm-1, ate omugerageranyo gw’okusaanawo kwa RC-LH116 ogupimiddwa ku 880 nm guli 3800±30 mM-1 cm-1.
UQ10 yapimibwa okusinziira ku nkola eri mu (52). Mu bufunze, HPLC eya reverse phase (RP-HPLC) yakolebwa nga tukozesa enkola ya Agilent 1200 HPLC. Saanuula nga 0.02 nmol za RC-LH116 oba RC-LH114-W mu 50μl za 50:50 methanol:chloroform ezirimu 0.02% (w/v) ferric chloride, era ofuyire Beckman Coulter Ultrasphere ODS 4.6 mm Esaanuuse mu 1 ml-1 min-1 ku 40°C mu HPLC ekizimbulukusa (80:20 methanol:2-propanol) ku mpagi ya ×25 cm. Kola isocratic elution mu HPLC solvent okulondoola okunyiga ku 275 nm (UQ10), 450 nm (carotenoids) ne 780 nm (BChl) okumala essaawa emu. Entiiko mu 275 nm chromatogram ku ddakiika 25.5 yagattibwa, nga temuli bikozesebwa birala byonna ebizuulibwa. Ekitundu ekigatta kikozesebwa okubala omuwendo gwa molar ogwa UQ10 oguggiddwamu nga tujuliza ekipimo ky’okupima ekyabalirirwa okuva mu kufuyira omutindo omulongoofu okuva ku 0 okutuuka ku 5.8 nmol (Ekifaananyi S14). Buli sampuli yeekenneenyezebwa mu mirundi esatu, era ensobi eyaloopebwa ekwatagana ne SD ya wakati.
Ekisengejjero ekirimu ekirungo kya RC-LH1 nga kirimu okunyiga kwa Qy okusinga obunene okwa 0.1 kyategekebwa nga kirimu 30 μM ekendeezeddwako cytochrome y’omutima gw’embalaasi c2 (Merck, UK) ne 0 okutuuka ku 50 μMUQ2 (Merck, UK). Sampuli ssatu eza 1-ml zategekebwa ku buli kigero kya UQ2 ne zifumbirwa ekiro kyonna mu nzikiza ku 4°C okukakasa nti zikwatagana bulungi n’ekizikiza nga tezinnaba kupima. Ekisengejjero kyatikkibwa mu OLIS RSM1000 modular spectrophotometer nga kirimu 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm wakati ne mm 0.6 outlet slits. Omusengejja ogw’okuyita mu buwanvu bwa nm 600 guteekebwa ku mulyango gwa ttanka y’ekitangaala eky’ekyokulabirako ne ttanka y’ekitangaala ekikubisaamu eky’okujuliza okuggyako ekitangaala eky’okusikirizibwa. Okunyiga kwalondoolebwa ku 550 nm nga obudde bw’okugatta bwa s 0.15. Ekitangaala ekisikiriza kifulumizibwa okuva mu 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) okuyita mu waya ya fiber optic ku maanyi ga 90% okuyita mu DC2200 controller (Thorlabs Ltd., UK) era kifulumizibwa ku nsibuko y’ekitangaala ku nkoona ya 90° of. Ekipimo ekipima kikontana n’endabirwamu okuzzaawo ekitangaala kyonna ekyali tekisooka kunyigibwa sampuli. Londoola okunyiga s 10 nga tekunnabaawo kwaka kwa s 50. Awo okunyiga kwayongera okulondoolebwa okumala s 60 mu nzikiza okwekenneenya ekigero quinolol ky’akendeeza ku cytochrome c23 + mu ngeri eyeetongodde (laba ekifaananyi S8 ku data embisi).
Data yakolebwa nga ekwatagana n’omutindo ogusookerwako ogw’ennyiriri mu s 0.5 okutuuka ku 10 (okusinziira ku bungi bwa UQ2) n’okugerageranya emiwendo gya sampuli zonsatule ku buli bungi bwa UQ2. Ekipimo kya RC-LH1 ekyabalirirwa omugerageranyo gw’okusaanawo ogukwatagana kyakozesebwa okukyusa omutindo mu bulungibwansi bw’okutabula, ne kiwandiikibwa mu Origin Pro 2019 (OriginLab, USA), ne kiteekebwa ku nkola ya Michaelis-Menten okuzuula emiwendo gya Km ne Kcat egyalabika.
Ku bipimo by’okunyiga okw’ekiseera ekitono, sampuli ya RC-LH1 yafukibwa okutuuka ku ~2μM mu IMAC buffer erimu 50 mM sodium ascorbate (Merck, USA) ne 0.4 mM Terbutin (Merck, USA). Ascorbic acid ekozesebwa nga omugabi wa obusannyalazo obusaddaaka, ate tert-butaclofen ekozesebwa nga ekiziyiza QB okukakasa nti omugabi wa RC omukulu asigala nga akendedde (kwe kugamba, si photooxidized) mu nkola yonna ey’okupima. Nga 3 ml za sampuli zigattibwa mu katoffaali akakyukakyuka aka custom (nga 0.1 m mu buwanvu, 350 RPM) nga kalina obuwanvu bw’ekkubo ery’amaaso erya mm 2 okukakasa nti sampuli eri mu kkubo lya layisi erina obudde obumala okukyusakyusa mu kizikiza wakati w’ebiwujjo by’okusikirizibwa. Kozesa ~100-fs laser pulses okugaziya Ti: Sapphire laser system (Spectra Physics, USA) okucamula sampuli ku 880 nm ku sipiidi y’okuddiŋŋana eya 1 kHz (20 nJ ku NIR oba 100 nJ ku Vis). Nga tonnakung’aanya data, sampuli giteeke mu kitangaala eky’okusikirizibwa okumala eddakiika nga 30. Okubikkulwa kijja kuleeta QA obutakola (kiyinzika okukendeeza QA omulundi gumu oba ebiri). Naye nsaba omanye nti enkola eno ekyusibwa kubanga oluvannyuma lw’ekiseera ekiwanvu eky’okutuukagana n’ekizikiza, RC ejja kudda mpola mu mirimu gya QA. Ekyuma ekipima ebifaananyi ekya Helios spectrometer (Ultrafast Systems, USA) kyakozesebwa okupima spektira ezitambula nga zirina obudde bw’okulwawo okuva ku -10 okutuuka ku 7000 ps . Kozesa pulogulaamu ya Surface Xplorer (Ultrafast Systems, USA) okuggyako ensengeka za data, olwo okugatta n’okuziteeka ku mutindo. Kozesa pulogulaamu ya CarpetView (Light Conversion Ltd., Lithuania) okukozesa ekibiina kya data ekigatta okufuna spektira ez’enjawulo ezikwatagana n’okuvunda, oba kozesa omulimu oguzingulula ebiraga ebingi n’okuddamu kw’ekintu okutuukagana n’enkulaakulana y’ensengekera y’obuwanvu bw’amayengo agamu mu Origin (OriginLab, USA).
Nga bwe kyayogeddwa waggulu (53), firimu ekola ekitangaala erimu LH1 complex etaliimu byombi RC ne peripheral LH2 antenna yategekebwa. Olususu lwafukibwa mu 20 mM tris (pH 8.0) oluvannyuma ne litikkibwa mu quartz cuvette nga erina ekkubo ly’amaaso erya mm 2. Layisi pulse ya 30nJ yakozesebwa okucamula sampuli ku 540 nm nga obudde bw’okulwawo bwa -10 okutuuka ku 7000 ps. Kola ku data set nga bwe kinyonyoddwa ku Rps. pal sampuli.
Olususu lwafuulibwa obukuta nga luyita mu kusengejja ku 150,000 RCF okumala essaawa 2 ku 4°C, n’oluvannyuma okunyiga kwalwo ku 880 nm ne kuddamu okusibwa mu 20 mM tris-HCl (pH 8.0) ne 200 mM NaCl. Saanuula oluwuzi ng’osika mpola mu 2% (w/v) β-DDM okumala essaawa 1 mu nzikiza ku 4°C. Sampuli yafukibwa mu 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) okutuuka ku puloteyina ya 2.5 mg ml-1 (Bio-Rad analysis). Okwongera okulongoosa kwakolebwa okuva mu nkola eyafulumizibwa emabegako (54), okutandika n’okufukirira puloteyina 50 μg mu muwendo gwonna ogwa 50 μl TEAB erimu 1% (w/v) sodium laurate (Merck, UK). Oluvannyuma lwa sonication okumala s 60, kyakendeezebwa ne 5 mM tris (2-carboxyethyl) phosphine (Merck, UK) ku 37 ° C okumala eddakiika 30. Ku S-alkylation, fumbira sampuli ne 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) era ogiteeke okuva mu 200 mM isopropanol stock solution okumala eddakiika 10 ku bbugumu erya bulijjo. Okugaaya obutoffaali (proteolytic digestion) kwakolebwa nga bongerako 2 μg trypsin/endoproteinase Lys-C mixture (Promega UK) ne kifumbirwa ku 37°C okumala essaawa 3. Ekirungo ekikola ku ttaka ekya laurate kyaggyibwamu nga kigattibwamu 50 μl ethyl acetate ne 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) ne kifuumuulwa okumala s 60. Okwawukana kwa phase kwatumbulwa nga tuyita mu centrifugation ku 15,700 RCF okumala eddakiika 5. Okusinziira ku nkola y’abakola, C18 spin column (Thermo Fisher Scientific, UK) yakozesebwa okusikiriza n’obwegendereza okuggyamu omunnyo mu kitundu ekya wansi ekirimu peptide. Oluvannyuma lw’okukala nga tuyita mu vacuum centrifugation, sampuli yasaanuusibwa mu 0.5% TFA ne 3% acetonitrile, era 500 ng yakeberebwa mu nanoflow RP chromatography nga egattibwako mass spectrometry nga tukozesa enkola parameters ezaayogerwako emabegako.
Kozesa MaxQuant v.1.5.3.30 (56) okuzuula puloteyina n’okugera obungi okunoonya Rps. ekifo eky’okuterekamu obutoffaali bw’obutoffaali obuyitibwa palustris proteome (www.uniprot.org/proteomes/UP000001426). Data ya mass spectrometry proteomics eterekeddwa mu ProteomeXchange Alliance okuyita mu tterekero ly’omukwanaganya wa PRIDE (http://proteomecentral.proteomexchange.org) wansi w’ekimanyisa dataset PXD020402.
Okusobola okwekenneenya nga bakozesa RPLC nga kugattibwako electrospray ionization mass spectrometry, RC-LH1 complex yategekebwa okuva mu wild-type Rps. Nga tukozesa enkola eyafulumizibwa emabegako (16), ekirungo kya puloteyina ekyakolebwa mu butoffaali bwa palustris kyali 2 mg ml-1 mu 20 mM Hepes (pH 7.8), 100 mM NaCl ne 0.03% (w/v) β- (Bio-Rad analysis) ) DDM. Okusinziira ku nkola y’omukozi, kozesa ekintu ekirongoosa ekya 2D (GE Healthcare, USA) okuggyamu puloteyina 10 μg ng’okozesa enkola y’okutonnyesa, era osaanuuse ekitonnyeze mu 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile ne 20% (v/v) amazzi. Microliters ttaano zaakeberebwa mu ngeri ya RPLC (Dionex RSLC) nga zigatta ku mass spectrometry (Maxis UHR-TOF, Bruker). Kozesa empagi ya MabPac 1.2×100 mm (Thermo Fisher Scientific, UK) okwawula ku 60°C ne 100μlmin -1, nga erina ekisengejjo kya 85% (v / v) ekizimbulukusa A [0.1% (v / v) FA ne 0.02% (V/v) TFA aqueous solution] okutuuka ku 85%(v/v) ekizimbulukusa B [0.1%(v/v) FA ne 0.02%(v/v) mu 90%(v/v) acetonitrile TFA] Nga tukozesa ensibuko ya ionization ey’amasannyalaze eya bulijjo n’ebipimo ebisookerwako okumala eddakiika ezisukka mu 60, ekipima ekipima ekinene kifuna 100 ku 2750 m/z (omugerageranyo gw’amasasi n’omusannyalazo). Nga tuyambibwako omukutu gwa ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), maapu y’ekisengejjo ky’amasasi ku subunits z’ekizibu.
Obutoffaali bwakuzibwa okumala essaawa 72 wansi wa 100 ml NF-low (10μMm-2 s-1), medium (30μMm-2 s-1) oba high (300μMm-2 s-1) light. M22 medium (M22 medium nga ammonium sulfate erekeddwawo ate sodium succinate n’akyusibwamu sodium acetate) mu ccupa ya 100 ml eriko sikulaapu (23). Mu nsengekera ttaano eza s 30, obululu bw’endabirwamu obwa micron 0.1 bwassibwako obululu ku mugerageranyo gw’obuzito bwa 1:1 okusobola okuwunyiriza obutoffaali ne bunyogozebwa ku ice okumala eddakiika 5. Ekintu ekitasaanuuka, obutoffaali obutamenyese n’obululu bw’endabirwamu byaggyibwawo nga biyita mu kusengejja ku 16,000 RCF okumala eddakiika 10 mu benchtop microcentrifuge. Olususu lwawulwa mu Ti 70.1 rotor nga 100,000 RCF mu 20 mM tris-HCl (pH 8.0) nga 40/15% (w/w) sucrose gradient okumala essaawa 10.
Nga bwe kyayogerwako mu mulimu gwaffe ogwasooka, immunodetection of the His tag ku PufW (16). Mu bufunze, ekizibu ky’omusingi ekirongooseddwa (11.8 nM) oba oluwuzi olulimu ekirungo kye kimu ekya RC (ekisalibwawo okuyitira mu oxidation okuggyako enjawulo ekendedde spectrum n’okukwatagana n’omugugu ku ggelu eriko amabala) mu 2x SDS loading buffer (Merck, UK) eyafukibwa emirundi ebiri. Puloteeni zino zaawulwa ku ggelu ya bis-tris NuPage eya 12% (Thermo Fisher Scientific, UK). Gelu yasiigibwa langi ya Coomassie Brilliant Blue (Bio-Rad, UK) okutikka n’okulaba ekitundu kya RC-L. Puloteeni eri ku ggelu eyookubiri yakyusibwa n’eteekebwa mu luwuzi lwa methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) okusobola okukebera obusimu obuziyiza endwadde. Olususu lwa PVDF lwazibikira mu 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 ne 5% (w / v) butto w’amata agafumbiddwa, n’oluvannyuma ne kifumbirwa n’ekiziyiza ekisookerwako ekiziyiza His (mu Dilute the antibody buffer [50 mM tris-HCl (pH 7.6), n’oluvannyuma ne kifumbirwa n’ekirungo ekiziyiza obuwuka obuziyiza endwadde (mu Dilute the antibody buffer [50 mM tris-HCl (pH 7.6),). 150 mM NaCl ne 0.05% (v/v) Tween-20] mu 1:1000 A190-114A, Bethyl Laboratories, USA) okumala essaawa 4. Oluvannyuma lw’okunaaba emirundi 3 okumala eddakiika 5 mu antibody buffer, oluwuzi lwagattibwa ne horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (diluted 1:10,000 mu antibody buffer) Fumbira okusobozesa okuzuula (eddakiika 5 oluvannyuma lw’okunaaba emirundi 3 mu antibody buffer) nga tukozesa WESTAR ETA C 2.0 chemiluminescence substrate (Cyanagen, Yitale) ne Amersham Imager 600 (GE Healthcare, Bungereza).
Nga tukuba ensaasaanya y’amaanyi ga buli ggelu eriko langi oba omulyango gw’okukebera obusimu obuziyiza endwadde, okugatta ekitundu wansi w’entikko n’okubala omugerageranyo gw’amaanyi ga RC-L (gel eriko langi) ne Protein-W (immunoassay), mu ImageJ (57) Process the image. Emigerageranyo gino gyakyusibwa ne gifuulibwa emigerageranyo gya molar nga tulowooza nti omugerageranyo gwa RC-L ne protein-W mu sampuli ya RC-LH114-W ennongoofu gwali 1:1 era ne tufuula data set yonna eya bulijjo okusinziira ku ekyo.
Okumanya ebikozesebwa eby’okugatta ku kiwandiiko kino, laba http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
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David J. K. 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
Ensengekera y’obulungi obw’amaanyi (high-resolution structure) ey’ekizibu ky’omutego gw’ekitangaala 1 mu kifo ky’ensengekera (reaction center) etuwa amagezi amapya ku nkyukakyuka ya quinone.
David J. K. 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
Ensengekera y’obulungi obw’amaanyi (high-resolution structure) ey’ekizibu ky’omutego gw’ekitangaala 1 mu kifo ky’ensengekera (reaction center) etuwa amagezi amapya ku nkyukakyuka ya quinone.
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