Fifia†Adrɛs si li fifia: OX11 0DE, UK, Diamond Xɔ, Harwell Dzɔdzɔmeŋutinunya kple Nu yeyewo dodo ɖe ŋgɔ ƒe Tsaɖibɔ, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Elektrɔnik Nu Gbagbewo ƒe Nɔnɔmetata Dɔwɔƒe.
Reaction center light-harvesting complex 1 (RC-LH1) ye nye keklẽŋusẽdɔlékui siwo ƒe amadede nye aŋutiɖiɖi ƒe akpa vevitɔ. Míeto RC-LH1 complex si tso Rhodopseudomonas palustris me ƒe cryo-electron microscopy structure eve vɛ. RC-LH114-W ƒe ƒuƒoƒoa ƒe 2.65-Å ƒe vovototodedeameme ƒe wɔwɔme nye subunit LH1 loops 14 siwo ƒo xlã RC, si protein W doa kplamatse, gake complex si me protein-W mele o ya nye RC ƒe wɔwɔme keŋkeŋ si ƒo xlã RC. Wotu 16 subunit LH1 ƒe ʋuƒo. Dɔwɔnu siawo tsɔtsɔ sɔ kple wo nɔewo naa gɔmesese deto le quinone ƒe ŋusẽkpɔɖeamedzi ŋu le RC-LH1 ƒe ƒuƒoƒoa me, siwo dometɔ aɖewoe nye nɔnɔme ƒe tɔtrɔ siwo womekpɔ tsã o ne wole quinone blam le RC QB teƒea, kpakple afisi kpeɖeŋutɔ quinone blana teƒewo le, si kpena ɖe wo ŋu Wotoa wo yia RC. W protein ƒe wɔwɔme tɔxɛa xea mɔ na LH1 ƒe ʋuƒoa ƒe nutsotso, si wɔnɛ be mɔ aɖe nɔa anyi si dzi woato ana quinone/quinolone ƒe asitɔtrɔ kabakaba.
Ŋusẽ si kekeli ƒe dɔwɔwɔ nana ate ŋu alé nu gbagbe siwo katã kloe le anyigba dzi la ɖe te, eye ŋutete gã aɖe le eŋu na ɣe ƒe nugbagbeŋutinunya. Togbɔ be dɔlékui siwo ƒe amadede nye aŋutiɖiɖi doa xexeame katã ƒe keklẽŋusẽ ƒe dɔwɔwɔ ɖe ŋgɔ hã la, woɖea ŋusẽ ƒe nɔnɔme vovovowo kple lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe ŋutete vovovowo hã fiana. Woate ŋu aƒo asa na keklẽŋusẽ ƒe dɔwɔwɔ eye woatsi abe dɔlékui siwo ɖua nu vovovowo ene le viviti me, woate ŋu aɖɔ nitrogen kple carbon dioxide ɖo, awɔ haidrodzin, eye woagblẽ atike ʋeʋẽewo dome (1-3). Be woate ŋu ana ŋusẽ nasu dɔ siawo wɔwɔ me la, ele be woatrɔ kekeli wòazu atikeŋusẽ kabakaba eye wòawɔ dɔ nyuie. Dɔ sia dzea egɔme ne kekeli-mɔ̃ ƒe antenna ƒe ƒuƒoƒoa xɔ kekeli eye wòtsɔ ŋusẽ si lé ɖe mɔ̃a me la yi ɖe nuwɔna ƒe teƒe (RC), si wɔnɛ be wodzea charge ƒe mama gɔme (4 – 7). Kekeli ƒe dɔwɔwɔ ƒe akpa vevitɔ le keklẽŋusẽdɔlékui siwo ƒe amadede nye aŋutiɖiɖi me la nye RC ƒomevi 2, si ŋu kekeli ƒe nuŋeŋe ƒe ƒuƒoƒo 1 (LH1) ƒo xlãe, si wɔ RC-LH1 ƒe nuto vevi la. LH1 nye αβ heterodimers siwo ƒo xlãe ƒe hatsotso aɖe, siwo dometɔ ɖesiaɖe blaa dɔlékui ƒe chlorophyll (BChl) a molecule eve kple carotenoid ɖeka alo eve (8-12). LH1 antenna bɔbɔetɔ kekeake nye αβ heterodimers 16 alo 17 siwo ƒo xlã RC (9-13) le loop si wotu me, gake le core complex bubuwo me la, transmembrane peptides doa kplamatse LH1 si ƒo xlãe ƒe yiyi ɖe edzi, To esia me doa quinol/quinone ƒe kaka ɖe ŋgɔ le RC kple cytochrome bc1 complex dome (11, 13-15) me. Numiemie si ƒe amadede nye aŋutiɖiɖi si nye Rhodopseudomonas (Rps.) nye kpɔɖeŋunu gbagbe si ate ŋu ase ŋusẽ kple elektrɔnik ƒe asitɔtrɔ si doa alɔ keklẽŋusẽ ƒe dɔwɔwɔ gɔme. Kristmas ƒe wɔwɔme gbãtɔ si nye Rps. Palustris RC-LH1 complex ƒe kpɔɖeŋue nye RC, si heterodimeric LH1 loop 15 ƒo xlãe, siwo protein si womenya o si woyɔna be “Protein W” (14) do kplamatse wo. Emegbe wode dzesi protein-W be enye RPA4402, si nye protein si ƒe lolome nye 10.5kDa si womede dzesii o si si transmembrane helices (TMH) etɔ̃ siwo wogblɔ ɖi le (16). Míeɖoe be míatrɔ ŋkɔ na rpa4402 domenyiŋusẽfianu si ŋlɔa protein W wòazu pufW be wòasɔ kple ŋkɔnya si wozãna na domenyiŋusẽfianu siwo ŋlɔa RC-L, M (pufL, pufM) kple LH1α, β (pufA, pufB) ƒe akpa suewo. Enyo be míade dzesii be protein-W le RC-LH1 ƒe 10% lɔƒo me ko, si ɖea Rps fiana. palustris wɔa RC-LH1 ƒe ƒuƒoƒo vovovo eve. Le afisia la, míegblɔ cryo-EM (cryo-EM) ƒe wɔwɔme siwo ƒe nɔnɔme deŋgɔ le nu vevi eve siwo me kɔ, ɖeka si me protein W kple 14 αβ heterodimers le, evelia si me protein W mele o kple 16 Heterodimer LH1 loop si wotu. Míaƒe wɔwɔme tsi tre ɖi na afɔɖeɖe ƒe tɔtrɔ le gɔmesese si le RC-LH1 complex of Rps ŋu. palustris, elabena míedzro vovototo ɖesiaɖe ƒe xexlẽme si sɔ me eye míaƒe tameɖoɖo kplikpaa si sɔ be míade peptide ɖesiaɖe kple amadede siwo wobla kple lipid kple quinones siwo do ƒome kplii la eme kɔte. Ne wotsɔ wɔwɔme siawo sɔ kple wo nɔewo la, woɖee fia be TMH protein-W etɔ̃ siwo womekpɔ le RC-LH1 ƒe ƒuƒoƒo bubu aɖeke me vaseɖe fifia o la wɔa quinone mɔ aɖe si naa quinone/quinolone ƒe asitɔtrɔ kabakaba. Wode dzesi ami kple quinone ƒe kadodoƒe geɖe siwo wodzra ɖo, eye míeɖe nɔnɔme ƒe tɔtrɔ yeye aɖe fia le quinone kple RC ƒe ƒoƒo ƒu megbe, si ate ŋu asɔ na fotoɖemɔ̃ II (PSII) RC na nugbagbevi siwo me oxygen le siwo ɖua foto. Míaƒe nusiwo míekpɔ na gɔmesese yeyewo le quinone/quinolone ƒe kadodo kple asitɔtrɔ ƒe ʋuʋu le RC-LH1 core complex si nye purple phototrophic bacteria me ŋu.
Be woate ŋu awɔ numekuku tsitotsito tso nusiwo wotsɔ wɔa nu eve siwo wokpɔ le Rps. palustris, míeɖea RC-LH1 ɖesiaɖe ɖe vovo to biochemical mɔnuwo dzi. Wokɔ protein W-manɔsitɔ ƒe ƒuƒoƒo (si woyɔna le afisia be ΔpufW) ŋu tso ƒome si me pufW domenyiŋusẽfianu mele o me (16), eye RC-LH1 ƒe ƒuƒoƒo ɖeka koe woate ŋu awɔ. Protein W-si me nu vovovowo le la tsoa ƒomevi aɖe gbɔ. Wotsɔa His ƒe dzesi si ƒe lolome nye 10x trɔa asi le protein W si le eƒe C-nuwuƒe ŋu, ale be woate ŋu atsɔ protein W-si le complex la akpe ɖe protein W si mebɔ o ƒe akpa gãtɔ ŋu nyuie to ga si meʋãna o me. Woɖea nusiwo me kɔ la ɖe vovo nyuie (16) Affinity Chromatography (IMAC).
Abe alesi woɖee fia le nɔnɔmetata 1 me ene la, RC sue etɔ̃ (RC-L, RC-M kple RC-H) si ŋu LH1 antenna ƒo xlãe le complex eveawo siaa me. 2.80-A ƒe wɔwɔme si le complex si me protein-W mele o la ɖe αβ heterodimers 16 fia, si wɔ LH1 loop si wotu si ƒo xlã RC keŋkeŋ, si woyɔna le afisia be RC-LH116 complex. 2.65Å ƒe wɔwɔme si le protein-W-si me ƒuƒoƒoa me la ƒe 14-heterodimer LH1 si protein-W do kplamatse, si woyɔna le afisia be RC-LH114-W.
(A kple B) Nusiwo wotsɔ wɔe ƒe anyigba ƒe nɔnɔmetata. (C kple D) Amadede siwo bla ɖe wo nɔewo ŋu siwo woɖena fiana le atikplɔwo me. . sphaeroides ƒe ƒuƒoƒo (13)]. Le LH1-α gome la, protein ƒe akpa suea ƒe amadede nyea ɣie; le LH1-β gome la, protein ƒe akpa suea ƒe amadede nyea blɔ; le protein-W gome la, protein la nyea dzĩ; le RC-H gome la, enye cyan; le RC-L gome la, enye Orange; na RC-M, si nye magenta. Wotsɔ atikplɔwo tsi tre ɖi na cofactors, amadede dzẽ tsi tre ɖi na BChl kple BPh a molecule, purple tsi tre ɖi na carotenoids, eye yellow tsi tre ɖi na UQ10 molecules. (G kple H) Protein-W ƒe dometsotso si le nuto si sɔ me le RC-LH114-W complex (G) kple RC-LH116 complex (H) ƒe nukpɔkpɔ si wodo ɖe ŋgɔ. Woɖea cofactors fiana le teƒe yɔyɔ ƒe nɔnɔme me, woɖea chelated quinone fiana le blɔ me. Wotsɔ fli blɔ si le (G) me de dzesi protein-W dometsotsoa, ​​eye wotsɔ fli yibɔ si le (H) me de dzesi do sue siwo me quinone/quinolol kaka ɖo le LH116 ƒe asigɛa dzi.
Nutata 1 (A kple B) fia RC si ƒo xlã LH1αβ heterodimers ƒe ƒuƒoƒo siwo le ʋuʋu alo esiwo wotu, siwo dometɔ ɖesiaɖe blaa BChl eve kple carotenoid ɖeka (Nɔnɔmetata 1, C kple D). Numekuku siwo wowɔ va yi ɖee fia be Rps ye nye LH1 ƒe ƒuƒoƒo. Le spirulina xanthin ƒe biosynthetic mɔ dzi la, carotenoids ƒe hatsotso vovovowo le lã ƒomevi siawo me ( 17 ). Gake spiropyrroxanthin ye nye carotenoid si xɔ aƒe ɖe eme eye eƒe kpekpeme dzea ame ŋu. Eyata míetiae be míawɔ spiroxanthin ƒe kpɔɖeŋu le LH1 ƒe kadodoƒewo katã. Alpha kple beta polypeptidewo nye TMH ɖekaɖeka siwo ƒe gotagome nuto siwo me lãkusi le kpuie (Nɔnɔmetata 1, A, B, E, kple F). Togbɔ be womede dzesi nusiwo susɔ 17 ƒe kpekpeme le C-nuwuƒea o hã la, woma alpha polypeptide la me tso Met1 va zu Ala46 le ƒuƒoƒo eveawo siaa me. Woɖe β polypeptide dzi kpɔtɔ tso Gly4 va zu Tyr52 le RC-LH116 me, eye tso Ser5 va zu Tyr52 le RC-LH114-W me. Womekpɔ N-nuwuƒe 3 alo 4 alo C-nuwuƒe ƒe nugbagbevi 13 ƒe kpekpeme aɖeke o (Nɔnɔmetata S1). Mass spectrometry numekuku le RC-LH1 complex si wotsaka si wodzra ɖo tso gbemelã ƒomevi la me ɖee fia be nuto si bu la nye heterologous cleavage of these peptides (Nɔnɔmetata S1 kple S2). Wokpɔ α-Met1 ƒe N-nuwuƒe ƒe wɔwɔme hã (f). Kukuɖenuŋua ɖee fia be α-peptide la nye nusiwo susɔ fMet1 vaseɖe Asp42/Ala46/Ala47/Ala50, eye β-peptide la nye nusiwo susɔ Ser2 vaseɖe Ala53, si wɔ ɖeka nyuie kple dzoxɔxɔ si bɔbɔ ƒe EM ƒe kpekpeme ƒe nɔnɔmetata.
α-His29 kple β-His36 ƒe ɖekawɔwɔ nana BChls ŋkume kple ŋkume; αβ heterodimer ɖesiaɖe ƒoa ƒu kple eƒe aƒelikawo wɔa ʋuƒo si le ʋuʋu ɖi (RC-LH114-W) alo ʋuƒo si wotu (RC-LH116) ƒo xlã RC The exciton coupled pigment array (Nɔnɔmetata 1, C kple D). Ne wotsɔe sɔ kple RC-LH114-W ƒe 877 nm band la, RC-LH116 ƒe 880 nm absorption red shift nye 3 nm (Nɔnɔmetata 2A). Ke hã, circular dichroism spectrum la sɔ kloe (Nɔnɔmetata 2B), si ɖee fia be togbɔ be vovototo si dze ƒã le loop siwo le ʋuʋu kple esiwo wotu dome hã la, BChls ƒe nutoa me ƒe nɔnɔme ɖi wo nɔewo ŋutɔ. Absorption redshift ate ŋu atso dzoxɔxɔ ƒe ʋuʋu si dzi ɖe kpɔtɔ kple liƒo si dzi ɖe edzi le loop si wotu dzi (18, 19), tɔtrɔ si va pigment coupling si loop si wotu gbɔ (20, 21), alo ŋusẽkpɔɖeamedzi eve siawo ƒe ƒuƒoƒo (11) gbɔ.
(A) Ultraviolet/visible/near-infrared absorption spectrum, si ƒe tawo wotsɔ woƒe amadede siwo sɔ kple wo nɔewo de dzesii eye wowɔ wo wòsɔ ɖe BPh ƒe taƒoƒo nu le 775 nm. (B) Kplɔ̃ŋuti dichroism spektrum normalized na BChl absorbance le 805 nm. (C kple D) Wotia ΔA spektrum tso RC-LH114-W complex (C) kple RC-LH116 complex (D) ƒe absorption spectra siwo wokpɔ le ɣeyiɣi me. Be woateŋu atsɔ wo asɔ kple wo nɔewo nyuie wu la, wowɔa spektrumwo katã wòsɔna ɖe ∆A si nye −A le 0.2 ps. (E) Cytochrome c2 ƒe oxidation ƒe agbɔsɔsɔme le keklẽŋusẽ dodo vɔ megbe le UQ2 ƒe agbɔsɔsɔ vovovowo ƒe anyinɔnɔ me (kpɔ Nɔnɔmetata S8 hena nyatakaka xoxowo). (F) Le lãmenugbagbevi siwo tsi le kekeli si ƒe ŋusẽ le sue, titina alo gã (10, 30 alo 300μMm-2 s-1, le wo nɔewo yome) te me la, protein W kple RC-L ƒe akpa sue siwo le complex si ŋu wokɔ me kple membrane ratio si woɖe ɖe vovo. Kpɔ protein ƒe agbɔsɔsɔ to SDS-polyacrylamide gel electrophoresis kple immunoassay dzi (kpɔ Nɔnɔmetata S9 hena nyatakaka xoxowo). Kpɔ xexlẽme si sɔ kple RC-LH114-W complex si ŋu wokɔ la ɖa. RC-L kple protein-W ƒe stoichiometric ratio le complex la me nye 1:1.
BChls siwo le nɔƒe 1 le RC-LH114-W ƒe αβ14 loop si gblẽ me (Nɔnɔmetata 1, A, C, kple E) te ɖe RC ƒe nunala gbãtɔ (P) ŋu wu 6.8Å wu BChls siwo sɔ le RC-LH116 me (Nɔnɔmetata. 1, B, D, kple F, kple Nɔnɔmetata S3); ke hã, ɣeyiɣi kpui aɖe ƒe absorption kinetics si le complex eveawo me ɖee fia be le RC-LH114-W kple RC-LH116 gome la, excitation energy transfer time constants tso LH1 yi RC nye 40 ±4 kple 44±3 ps (Nɔnɔmetata 2). , C kple D, Nɔnɔmetata S4 kple Tabla S2). Vovototo gã aɖeke hã mele elektrɔnikmɔ̃ dzi tsɔtsɔ yi RC me o (Nɔnɔmetata S5 kple kpeɖeŋutɔ nuŋɔŋlɔ siwo do ƒome kplii). Míesusu be ŋusẽ ƒe tsɔtsɔ yi teƒe bubu ƒe ɣeyiɣi ƒe sɔsɔ kplikplikpli si le LH1 kple RC-P dome la tso didiƒe, dzogoe kple ŋusẽ si ate ŋu anɔ BChl akpa gãtɔ si le LH1 ƒe ʋuƒo eveawo me ƒe sɔsɔ gbɔ. Edze abe LH1 ŋusẽ ƒe nɔnɔme me dzodzro be woaɖo didiƒe suetɔ kekeake gbɔ mele kabakaba wu ŋusẽ ƒe tsɔtsɔ tẽ tso teƒe siwo mesɔ o yi RC o ene. LH1 ƒe ʋuƒo si le ʋuʋu ɖi le RC-LH114-W me hã ate ŋu ato dzoxɔxɔ ƒe ʋuʋu si mehiã o me le dzoxɔxɔ ƒe nɔnɔme siwo mebɔ o me hena xɔtuɖaŋu me dzodzro, eye αβ14 ƒe asigɛ ƒe nɔnɔme didi wu le xɔme ƒe dzoxɔxɔ me tso βBChls ƒe amadede ƒe didime le RC 1 ƒe nɔƒe.
BChl 32 kple carotenoid 16 ye le RC-LH116 ƒe ƒuƒoƒoa me, eye eƒe ɖoɖo bliboa sɔ kple esi wokpɔ tso Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 ƒomevi ( PDB ID 7C9R) (12) kple amadede dzẽ me ƒumegbewo (Blc.viridis) (PDB ID 6ET5) (10). Le ɖoɖowɔwɔ vɔ megbe la, vovototo suesuesuewo koe wokpɔ le αβ heterodimers ƒe nɔƒewo, vevietɔ 1-5, 15, kple 16 (Nɔnɔmetata S6). Protein-W ƒe anyinɔnɔ kpɔa ŋusẽ gã aɖe ɖe LH1 ƒe wɔwɔme dzi. Eƒe TMH etɔ̃awo do ƒome kple ka kpuiwo, eye N-nuwuƒea le complex la ƒe lumen ƒe akpa dzi eye C-nuwuƒea le cytoplasmic ƒe akpa dzi (Nɔnɔmetata 1A kple 3, A vaseɖe D). Protein-W ƒe akpa gãtɔ melɔ̃a tsi o (Nɔnɔmetata 3B), eye TMH2 kple TMH3 wɔa dɔ kple LH1αβ-14 tsɔ wɔa transmembrane ƒe anyigba (Nɔnɔmetata 3, B kple E vaseɖe G). Phe, Leu kple Val ƒe nugbagbevi siwo susɔ le transmembrane nuto me koŋue le ƒomedodoa me. Wotsɔa amino acid siwo me tsi mele o kple αβ-14 amadedewo ƒoa ƒu ɖe nusiwo susɔ siawo ŋu. Polar ƒe nugbagbevi aɖewo hã kpena ɖe kadodoa ŋu, siwo dometɔ aɖewoe nye haidrodzin ƒe kadodo si le W-Thr68 kple β-Trp42 dome le do sesẽa ƒe anyime (Nɔnɔmetata 3, F kple G). Le lãmenugbagbeviwo ƒe anyime la, Gln34 te ɖe αβ-14 carotenoids ƒe keto ƒuƒoƒoa ŋu. Tsɔ kpe ɖe eŋu la, woɖe n-dodecyl β-d-maltoside (β-DDM) molecule la ɖa, eye eƒe asike si me tsi mele o la keke ta ɖo protein-W kple αβ-14 dome domedome, eye lipid ƒe asike ate ŋu anɔ ŋutilãa me. Míede dzesii hã be protein W kple RCH ƒe C-nuwuƒe ƒe vovototodedeameme nutowo te ɖe wo nɔewo ŋu ŋutɔ, gake womele kadodo tɔxɛwo wɔwɔ ƒe ŋutete me o (Nɔnɔmetata 1, A kple E). Gake kadodo ate ŋu anɔ protein eve siawo ƒe C-nuwuƒe amino acid siwo womekpɔ ta na o me, si ate ŋu ana mɔnu aɖe si dzi woato axɔ protein-W le RC-LH114-W ƒe ƒuƒoƒoa ƒe ƒuƒoƒo me.
(A) Protein-W, si dze ŋgɔ ƒomedodo kple LH1αβ14 le nutata ƒe nɔnɔme me la, ƒe axadzikɔsɔkɔsɔ si le abe atikplɔ ene (dzẽ), si woɖe fia le elektrikŋusẽ ƒe ŋutete ƒe nɔnɔmetata ƒe akpa aɖe (anyigba si ƒe amadede nye aŋutiɖiɖi si me kɔ si ƒe nɔnɔme nye 0.13). (B) Protein-W si wotsɔ amadede si me tsi mele o tsi tre ɖi na. Woɖea teƒe siwo polar kple charged fiana le cyan me, woɖea teƒe siwo tsi melɔ̃na o fiana le ɣie me, eye woɖea teƒe siwo tsi melɔ̃na o fiana le amadede aŋutiɖiɖi me. (C kple D) Protein-W si woɖe fia le nutata me, eƒe mɔfiame sɔ kple esi le (A) (C), eye wòtrɔ 180° (D). Le nɔƒe si le ɖoɖo si nu wole nu la, nusiwo susɔ siwo woate ŋu ade vovototo wo dome la xɔa dziŋgɔli ƒe amadede ƒe ɖoɖo, afisi N-nuwuƒe nye blɔ eye C-nuwuƒe nye dzĩ. (E) Protein-W le nukpɔkpɔ ma ke me abe alesi wòle le (A) me ene, eye wotsɔ atikplɔ siwo ŋu dzesiwo kpe ɖo la tsi tre ɖi na nusiwo susɔ le protein-W:LH1 ƒe domedome. (F) Wotrɔa protein-W 90° ne wotsɔe sɔ kple (E) kple LH1αβ14 le nutata ƒe nɔnɔmetata me, eye ne wotsɔe sɔ kple ƒomedodo ƒe susɔe siwo le bar ƒe nɔnɔmetata me. Wodea dzesi nusiwo susɔ siwo le beta polypeptide la me siwo le xɔxlɔ̃m. Woɖe cofactor la fia abe bar si sɔ kple nɔnɔmetata 1 ƒe amadede ene, woɖe β-DDM si gblẽ la fia le amadede ɣi me, eye woɖe oxygen la fia le amadede dzĩ me. (G) Wotrɔa nukpɔkpɔ si le (F) me 180°, eye alpha polypeptide si wode dzesii la ƒe nugbagbevi siwo dze ƒã la le eme.
Protein-W xɔna ɖe αβ heterodimer teƒe (si nye 15 lia le Nɔnɔmetata 1F me), si wɔnɛ be wòxea mɔ na loop ƒe nutsotso eye wòtrɔa αβ heterodimer etɔ̃ gbãtɔwo. Wode dzesii be αβ-1 heterodimer gbãtɔ ƒe dzogoe si sɔ gbɔ wu ne wotsɔe sɔ kple film normal la nye 25° va ɖo 29° (Nɔnɔmetata 1, A kple E), si wowɔ to αβ-1 ƒe 2° va ɖo 8° ƒe ŋgɔgbedede le RC A sharp contrast-LH116 me (Nɔnɔmetata 1, B kple F). Heterodimers evelia kple etɔ̃lia le 12° va ɖo 22° kple 5° va ɖo 10°, le wo nɔewo yome. Le RC ƒe steric mɔxenu ta la, αβ-1 ƒe tilt melɔ αβ eve evelia (si sɔ kple αβ 16 lia le Nɔnɔmetata 1F me) ɖe eme o, si wɔnɛ be dometsotso si me kɔ le LH1 ƒe asigɛa me (Nɔnɔmetata 1, A kple E) . Le αβ heterodimers eve ƒe anyimanɔmanɔ, si kpe ɖe BChl ene kple carotenoid eve ƒe bu ŋu ta la, carotenoids dometɔ aɖeke mebla ɖe αβ-1 ƒe akpa sue si wotro la ŋu o, si wɔnɛ be LH114-W ƒe asigɛ si me carotenoid 13 Vegetarian kple BChls 28 le la dona. Nutoa me ƒe vovototodedeameme ƒe akɔntabubu siwo le ƒuƒoƒo eve siwo le αβ1 va ɖo 7 nutoawo me la le ʋɛ wu LH1 ƒe ʋuƒo mamlɛawo tɔ, si ate ŋu aɖe LH1 ƒe akpa sue si te ɖe RC QB teƒea ŋu ƒe plasticity si le dzɔdzɔme nu afia (Nɔnɔmetata 4) .
Woɖe RC-LH114-W (A kple B) kple RC-LH116 (C kple D) ƒe nɔnɔmetatawo fia tso dziƒokpɔkpɔ/axadzikpɔkpɔ ɖeka ma ke (A kple B) (A kple C) kple do ƒe anyigba si le Fig. 1. (B kple D). Woɖe safui siwo le amadede vovovowo me la fia le ɖusime.
Nu vevi bubu si ɖe dzesi le nu vevi si ƒe stoichiometric ratio nye 1:14 koe nye Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Gake protein W kple PufX mewɔ ɖeka si dze ƒã o, eye wokpɔa ŋusẽ gã aɖe ɖe woƒe LH1 ƒe wɔwɔmewo dzi. PufX nye TMH ɖeka si si N-nuwuƒe ƒe lãmenugbagbeviwo ƒe domenyiŋusẽfianu le si wɔa dɔ kple RC-H ƒe akpa suea ƒe lãmenugbagbeviwo ƒe akpa (13) le teƒe si sɔ kple Rps. palustris LH116αβ-16 ƒe ʋukawo. PufX wɔa mɔnu na quinone/quinolone ƒe asitɔtrɔ le RC-LH1 kple cytochrome bcl complex dome eye wòle Rba katã me. sphaeroides ƒe nu veviwo ƒe ƒuƒoƒo (13). Togbɔ be monomer-monomer ƒe ƒomedodo le Rba me hã la. Sphaeroides RC-LH1-PufX dimer le protein W ƒe kadodoƒe le RC-LH114-W me, eye dometsotso si PufX kple protein-W hena vɛ la le teƒe si sɔ (Nɔnɔmetata S7A). Dodokpɔ si le RC-LH114-W me hã sɔ kple Pseudomonas rosea LH1 ƒe quinone mɔnu (8) si wosusu, si peptide siwo medo ƒome kple protein W alo PufX o wɔ (Nɔnɔmetata S7B). Tsɔ kpe ɖe eŋu la, quinone-mɔ̃ si le Blc. LH1 si ƒe amadede nye emerald si wowɔ to γ ​​ƒe akpa sue ɖeka (7) ɖeɖeɖa me la le teƒe ma tɔgbe (Nɔnɔmetata S7C). Togbɔ be protein vovovowoe nye domenɔlawo hã la, edze abe quinone/quinolol mɔ siawo ƒe dzedzeme le teƒe ɖeka le RC-LH1 ƒe ƒuƒoƒoa me nye nɔnɔmetɔtrɔ si wɔ ɖeka ƒe kpɔɖeŋu ene, si ɖee fia be dometsotso si protein W hena vɛ ate ŋu awɔ dɔ abe quinone mɔ ene.
Dodokpɔ si le LH114-W ƒe ʋuƒoa me ɖea mɔ be woawɔ lãkusi ƒe nuto si yia edzi le RC-LH114-W ƒe ƒuƒoƒoa ƒe ememe teƒe kple lãkusi gã la dome (Nɔnɔmetata 1G), tsɔ wu be woatsɔ domenyiŋusẽfianu eveawo aƒo ƒu to protein ƒe ʋuʋudedi me abe alesi wòle le proteinwo me ene. RC-LH116 ƒe ƒuƒoƒoa sɔ kple Tch si wotu. Nusi le abe aŋe ene (22) (Nɔnɔmetata 1H). Esi wònye be quinone ƒe kaka to lãkusia me kabakaba wu kaka to protein ƒe mɔ xaxɛa dzi ta la, LH114-W ƒe ka si le ʋuʋu ɖi ate ŋu aɖe mɔ na RC ƒe tɔtrɔ kabakaba wu LH116 ka si wotu, eye quinone ƒe kaka ɖe RC me ate ŋu axe mɔ ɖe enu wu. Be míadoe kpɔ be protein W kpɔa ŋusẽ ɖe quinones ƒe tɔtrɔ to RC dzi hã la, míewɔ cytochrome oxidation dodokpɔ le ubiquinone 2 (UQ2) (si nye dzɔdzɔme UQ10 ƒe nɔnɔmetata si ƒe asike le kpuie wu si ƒe isoprene) ƒe agbɔsɔsɔ aɖe dzi (Nɔnɔmetata 2E). Togbɔ be chelated quinone ƒe anyinɔnɔ xea mɔ na Michaelis ƒe nɔnɔme si dze abe ɖe wòle tɔtrɔm ene ƒe nyanya pɛpɛpɛ (RC-LH114-W kple RC-LH116 sɔ na 0.2±0.1μM kple 0.5±0.2μM, le wo nɔewo yome), RC-LH114-W ƒe agbɔsɔsɔ si sɔ gbɔ wu ( 4.6±0.2 e-RC-1 s-1) nye 28±5% lolo wu RC-LH116 (3.6±0.1 e-RC-1 s-1).
Míebu akɔnta le gɔmedzedzea me be protein-W le abe 10% ene le core complex (16); le afisia la, lãmenugbagbevi siwo me kekeli mele o, esiwo me kekeli le titina, kple esiwo me kekeli lolo le ƒe tsitsi ƒe lãmenugbagbeviwo ƒe nɔƒe nye 15±0.6%, 11±1% kple 0.9±0.5, le wo nɔewo yome % (Nɔnɔmetata 2F). Agbɔsɔsɔme tsɔtsɔ sɔ kple mass spectrometry ɖee fia be histidine tag tsɔtsɔ kpe ɖe eŋu meɖe protein-W ƒe agbɔsɔsɔ si sɔ dzi kpɔtɔ ne wotsɔe sɔ kple gbemelã ƒomeviwo (P = 0.59), eyata agbɔsɔsɔ siawo menye protein-W si wogbugbɔ trɔ ƒe asinudɔwɔwɔ o (Nɔnɔmetata S10). Gake protein-W ƒe anyimanɔmanɔ sue sia le RC-LH1 ƒe ƒuƒoƒoa me ate ŋu ana RC aɖewo natrɔ kabakaba, si ana quinone/quinolone ƒe asitɔtrɔ blewu le RC-LH116 ƒe ƒuƒoƒoa me naɖe akpɔtɔ. Míede dzesii be kekeli ƒe agbɔsɔsɔ si sɔ gbɔ la mewɔ ɖeka kple transcriptomics ƒe nyatakaka siwo woxɔ nyitsɔ laa o, si ɖee fia be pufW domenyiŋusẽfianu ƒe ɖeɖefia dzina ɖe edzi le kekeli sesẽ te (Nɔnɔmetata S11) (23). Vovototo si le pufW ƒe nuŋɔŋlɔ kple protein-W ƒe gege ɖe RC-LH1 ƒe ƒuƒoƒoa me dome tɔtɔa ame eye ate ŋu aɖe protein la ƒe ɖoɖowɔwɔ sesẽa afia.
Le RC-LH114-W me la, woma cardiolipin (CDL) 6, phosphatidylcholine (POPC) 7, phosphatidylglycerol (POPG) 1 kple β-DDM molecule 29 eye wowɔa kpɔɖeŋu le eme CDL 6, POPC 24, POPG 2 kple βDDM 12. RC-LH116 (Nɔnɔmetata 5, A kple B). Le wɔwɔme eve siawo me la, CDL le cytoplasmic ƒe akpa si le complex la me kloe, gake POPC, POPG kple β-DDM ya le luminal ƒe akpa gãtɔ. Woɖe ami kple atike si wotsɔna klɔa nu ƒe molecule eve ɖe vovo le αβ-1 va ɖo αβ-6 nuto si le RC-LH114-W complex (Nɔnɔmetata 5A), eye woɖe atɔ̃ ɖe vovo le RC-LH116 ƒe nuto si sɔ me (Nɔnɔmetata 5B ). Wokpɔ ami geɖe wu le nusiwo le ƒuƒoƒoa ƒe akpa kemɛ, vevietɔ CDL, siwo ƒo ƒu ɖe RC kple αβ-7 dome va ɖo αβ-13 (Nɔnɔmetata 5, A kple B). Lipid kple atike siwo wotsɔ klɔa nu bubu siwo ƒe wɔwɔme me kɔ la le LH1 ƒe asigɛa godo, eye acyl kɔsɔkɔsɔ siwo me kɔ nyuie la keke ta le LH1 ƒe akpa suewo dome, si wotsɔ ŋkɔ na le ɣeyiɣi aɖe me be β-DDM le RC-LH114-W me, eye woɖe egɔme be β-DDM le RC A β-DDM kple POPC-LH116 ƒe tsakatsaka me. Chelating lipids kple detergents ƒe nɔƒe mawo tɔgbe le míaƒe wɔwɔme me ɖee fia be wonye kadodoƒe siwo sɔ le ŋutilã me (Nɔnɔmetata S12A). Molekyul siwo sɔ ƒe nɔƒewo le Tch me hã sɔ nyuie. Fafa kple Trv. Ƒomevi 970 RC-LH1s (Nɔnɔmetata S12, B vaseɖe E) (9, 12) kple haidrodzin-kadodo ƒe susɔe siwo le lipid ta ƒe ƒuƒoƒoa me ɖee fia be wokpɔ wo ta nyuie ŋutɔ le tsiatretsitsi ƒe ɖoɖowɔwɔ me (Nɔnɔmetata S13), si fia be Conserved CDL si blana ɖe RC (24), teƒe siawo ate ŋu adzra ɖo le RC-LH1 ƒe ƒuƒoƒoa me.
. UQ si bla ɖe RC QA kple QB teƒewo nye ɣie, gake UQ si ɖe eɖokui ɖe aga ya nye blɔ. (C kple D) Nukpɔsusu siwo le (A) kple (B) si, eye woɖe ami siwo le eme ɖa. (E vaseɖe G) Q1(E), Q2(F) kple Q3(G) ƒe nukpɔkpɔ si lolo tso RC-LH116 me, kple axadzikɔsɔkɔsɔ siwo kpɔa ŋusẽ ɖe wo nɔewo dzi. Woɖe haidrodzin ƒe kadodoawo fia abe fli yibɔ siwo wotso ene.
Le RC-LH116 me la, RC QA kple QB UQ siaa, siwo kpɔa gome le elektrɔnik ƒe asitɔtrɔ me le charge mama ƒe ɖoɖoa me la, gblẽna le woƒe kadodoƒewo. Ke hã, le RC-LH114-W me la, womekpɔ QB quinone gbɔ o eye woaƒo nu tso eŋu tsitotsito le ete. Tsɔ kpe ɖe QA kple QB quinones ŋu la, woma UQ molecule eve siwo me chelated (siwo le RC kple LH1 ƒe asigɛwo dome) ɖe RC-LH114-W ƒe wɔwɔme me le woƒe ta ƒe ƒuƒoƒo siwo wokpɔ nyuie (siwo le Q1 kple Q2 me, le wo nɔewo yome) nu. teƒe gbadza). Nɔnɔmetata 5C). Wotsɔ isoprene ƒe akpa eve de asi na Q1, eye density map la kpɔa isoprene ƒe asike 10 bliboa gbɔ le Q2 me. Le RC-LH116 ƒe wɔwɔme me la, wokpɔ UQ10 molecule etɔ̃ siwo me chelated le (Q1 vaseɖe Q3, Nɔnɔmetata 5D) gbɔ, eye moleculeawo katã ƒe kpekpeme dze ƒã le asike bliboa me (Nɔnɔmetata 5, D vaseɖe G). Le xɔtuɖoɖo eveawo me la, Q1 kple Q2 ƒe quinone ta ƒuƒoƒoawo ƒe nɔƒewo sɔ nyuie ŋutɔ (Nɔnɔmetata S12F), eye RC koe wowɔa dɔ kple wo nɔewo. Q1 le RC-LH114-W ƒe W dometsotso ƒe mɔnu (Nɔnɔmetata 1G kple 5, C, D kple E), eye Q2 le teƒe si te ɖe QB ƒe kadodoƒe ŋu (Nɔnɔmetata 5, C, D) kple F). L-Trp143 kple L-Trp269 ƒe nugbagbevi siwo wodzra ɖo la te ɖe Q1 kple Q2 ŋu ŋutɔ eye wonaa π-stacking ƒe kadodo siwo ate ŋu adzɔ (Nɔnɔmetata 5, E kple F, kple Nɔnɔmetata S12). L-Gln88, si didi tso Q1 ƒe oxygen si le didiƒe gbɔ 3.0 Å, naa haidrodzin ƒe kadodo sesẽ aɖe (Nɔnɔmetata 5E); wodzraa nu susɔe sia ɖo le RCwo katã me negbe ƒomedodo si le didiƒe wu koe mele eme o (Nɔnɔmetata S13). L-Ser91 nye esi wotsɔ ɖɔ li Thr le mɔ si me woɖea mɔ ɖe nu ŋu le le RC bubu akpa gãtɔ me (Nɔnɔmetata S13), enye Angstrom 3.8 tso methyl oxygen si le Q1 me, eye ate ŋu ana haidrodzin ƒe kadodo siwo gbɔdzɔ (Nɔnɔmetata 5E). Edze abe kadodo aɖeke koŋ mele Q3 si o ene, gake ele nuto si me tsi mele o si le RC-M ƒe akpa sue kple LH1-α ƒe akpa sue 5 va ɖo 6 dome (Nɔnɔmetata 5, D kple G). Wokpɔ Q1, Q2 kple Q3 alo chelated quinones siwo te ɖe wo ŋu hã gbɔ le Tch. Fafa, Trv. Ƒomevi 970 kple Blc. Iris ƒe wɔwɔme (9, 10, 12) fia asi kpeɖeŋutɔ quinone ƒe kadodoƒe si wodzra ɖo le RC-LH1 ƒe ƒuƒoƒoa me (Nɔnɔmetata S12G). UQ atɔ̃ siwo gblẽ le RC-LH116 me wɔ ɖeka nyuie kple 5.8±0.7 si le complex ɖesiaɖe si wokpɔ to high performance liquid chromatography (HPLC) me, esime UQ etɔ̃ siwo gblẽ le RC-LH114-W me le ʋɛ wu The measured value of 6.2±0.3 (Fig. S14) indicates that there are unresolved UQ molecules in the structure.
TMH atɔ̃ le pseudo-symmetric L kple M polypeptide ɖesiaɖe me eye wowɔa heterodimer si ƒoa BChl dimer ɖeka, BChl monomers eve, bacteriophage (BPh) monomers eve, kple non- Heme iron ɖeka kple UQ10 molecule ɖeka alo eve nu ƒu. To haidrodzin ƒe kadodowo ƒe anyinɔnɔ le ketone ƒuƒoƒo si le nuwuƒe kple eƒe ƒuƒoƒo si wonya le Rps me me la, wotsɔa carotenoids dea M-subunit, si woyɔna be cis-3,4-dehydroorhodopin me. Lã ƒomeviwo (25). Wotsɔa TMH ɖeka léa RC-H ƒe gotagome lãkusi ƒe akpa ɖe lãkusia ŋu. RC ƒe wɔwɔme bliboa sɔ kple lã ƒomevi siwo do ƒome kplii (abe Rba ene) ƒe akpa sue etɔ̃ siwo nye RC. sphaeroides (PDB ID: 3I4D) ƒe ƒuƒoƒo. BChl kple BPh ƒe macrocycles, carotenoid backbone kple non-heme iron ƒoa ƒu ɖe xɔtuɖoɖo siawo ƒe resolution ƒe didime me, abe alesi UQ10 ta ƒuƒoƒo si le QA teƒea kple QB quinone le RC-LH116 hã wɔe ene (Nɔnɔmetata S15).
RC xɔtuɖoɖo eve siwo ƒe anyinɔnɔ kple QB teƒea ƒe amedzidzedze ƒe agbɔsɔsɔme to vovo na mɔnukpɔkpɔ yeye be woalé ŋku ɖe nɔnɔme ƒe tɔtrɔ siwo nɔa anyi ɖaa siwo kpe ɖe QB quinone ƒe kadodo ŋu la ŋu. Le RC-LH116 ƒe ƒuƒoƒoa me la, QB quinone le “teƒe si wobla bliboe” (26), gake QB quinone mele RC-LH114-W ƒe mama me o. QB quinone aɖeke mele RC-LH114-W me o, si wɔ nuku elabena complex la le dɔ wɔm, wu RC-LH116 complex si me structurally resolved QB quinone le. Togbɔ be LH1 ƒe asigɛ eveawo wɔa chelate abe quinones ade ene hã la, atɔ̃ ƒe wɔwɔme me kɔ le RC-LH116 ƒe asigɛ si wotu me, evɔ etɔ̃ koe ƒe wɔwɔme seɖoƒe li na le RC-LH114-W ƒe asigɛ si le ʋuʋu ɖi me . Dɔwɔɖoɖo ƒe kuxi sia si dzi ɖe edzi ate ŋu aɖe RC-LH114-W QB teƒewo ɖɔliɖɔli kabakaba, quinone ƒe ʋuʋu kabakaba le complex la me, kple alesi wòate ŋu adzɔe be woatso LH1 ƒe ʋuƒoa dzi ɖe edzi afia. Míeɖo aɖaŋu be UQ ƒe anyimanɔmanɔ le RC-LH114-W ƒe RC QB teƒe ate ŋu atso nusi sesẽ wu eye wòwɔa dɔ wu gbɔ, eye wotu tsikpe ɖe RC-LH114-W ƒe QB teƒe enumake le UQ ƒe tɔtrɔ me Afɔɖeɖe tɔxɛa (wotu QB teƒea ƒe mɔnu) ɖea dɔwɔwɔ sia ƒe nɔnɔme fiana.
QB manɔmee la, L-Phe217 ƒe tɔtrɔ si kpe ɖe eŋu yi teƒe si mewɔ ɖeka kple UQ10 ƒe kadodo o, elabena ana teƒe ƒe ƒoƒo kple asike ƒe isoprene unit gbãtɔ (Nɔnɔmetata 6A). Tsɔ kpe ɖe eŋu la, tɔtrɔ vevi siwo dze ƒã le nɔnɔme me la dze ƒã, vevietɔ helix de (helix kpui si le loop si le TMH D kple E dome) afisi woɖea L-Phe217 yia QB ƒe kadodokotokua me kple L-Tyr223 ƒe tɔtrɔ (Nɔnɔmetata 6A ) Be woagbã haidrodzin ƒe kadodo kple M-Asp45 ƒe xɔtuɖoɖoa eye woatu QB ƒe kadodoƒea ƒe mɔnu (Nɔnɔmetata 6B). Helix de pivots le eƒe gɔme, L-Ser209 ƒe Cα trɔna 0.33Å, esime L-Val221Cα trɔna 3.52Å. Tɔtrɔ aɖeke mele TMH D kple E, siwo woate ŋu atsɔ aɖo wo nɔewo dzi le xɔtunu eveawo siaa me o (Nɔnɔmetata 6A). Le alesi míenya nu la, esiae nye xɔtuɖoɖo gbãtɔ le dzɔdzɔme RC me si tu QB teƒea. Ne wotsɔe sɔ kple wɔwɔme bliboa (QB-bla) ɖee fia be hafi woaɖe quinone la dzi akpɔtɔ la, ehiã be woawɔ tɔtrɔ le eƒe nɔnɔme ŋu hafi wòage ɖe quinone la me. L-Phe217 trɔna tsɔ wɔa π-stacking kadodo kple quinone ta ƒuƒoƒoa, eye helix la trɔna yia gota, si wɔnɛ be L-Gly222 ƒe ƒunukpeƒe kple L-Tyr223 ƒe axadzikɔsɔkɔsɔa wɔa haidrodzin kadodo ƒe kadodo si me haidrodzin kadodo ƒe wɔwɔme li ke le (Nɔnɔmetata 6, A kple C).
(A) Hologram (L kɔsɔkɔsɔ, orange/M kɔsɔkɔsɔ, magenta) kple apo (gray) ƒe wɔwɔme ƒe nutata si ƒo xlã wo nɔewo, si me woɖea nu vevi siwo susɔ la fiana le nɔnɔmetata si le abe atikplɔ ene ƒe nɔnɔme me. Wotsɔa fli ɣi aɖe tsi tre ɖi na UQ10. Fli si dzi woŋlɔ nu ɖo la fia haidrodzin ƒe kadodo siwo wowɔ le xɔ bliboa me. (B kple C) Apolipoprotein kple asigɛ bliboa ƒe wɔwɔme ƒe gotagome nɔnɔmetata, si te gbe ɖe axadzi kɔsɔkɔsɔ oxygen si le L-Phe217 le blɔ kple L-Tyr223 le dzĩ me, ɖe wo nɔewo yome. L ƒe akpa suea nye aŋutiɖiɖi; M kple H ƒe akpa sueawo mekpɔa amadede o. (D kple E) Apolipoprotein (D) kple blibo (E) RC QB teƒewo [amadede si wotsɔ (A) wɔe] kple Thermophilus thermophilus PSII (amadede dzẽ, blɔ kple plastic quinone; PDB ID: 3WU2) Wɔ ɖeka (58).
Le mɔ si womele mɔ kpɔm na o nu la, togbɔ be RC siwo me QB mele o ƒe wɔwɔme geɖe li siwo me LH1 mele o hã la, womegblɔ tɔtrɔ siwo wokpɔ le nɔnɔme ƒe nɔnɔme me le numekuku sia me o. Esiawo dometɔ aɖewoe nye QB ƒe tsɔtsrɔ̃ ƒe ɖoɖo si tso Blc. viridis (PDB ID: 3PRC) (27), Tch. Ƒe 1999 ƒe ɣleti gbãtɔ me. tepidum (PDB ID: 1EYS) (28) kple Rba. sphaeroides (PDB ID: 1OGV) (29), siwo katã sɔ kple woƒe QB ƒe wɔwɔme bliboa kloe. Ne wolé ŋku ɖe 3PRC ŋu nyuie la, wokpɔe be LDAO (Lauryl Dimethyl Amine Oxide) ƒe atike si wotsɔna klɔa nu ƒe moleculewo blana le QB ƒe nɔnɔme ƒe mɔnu, si ate ŋu axe mɔ ɖe gbugbɔgaɖoɖo ɖe nɔnɔme si wotu nu. Togbɔ be LDAO megblẽna le teƒe ɖeka le 1EYS alo 1OGV me o hã la, wozãa atike ɖeka ma ke tsɔ dzraa RC siawo ɖo eye le esia ta woate ŋu awɔ dɔ ma ke. Rba ƒe kristalo ƒe wɔwɔme. Edze abe Sphaeroides RC si wotsɔ kristalo wɔ ɖekae kple cytochrome c2 (PDB ID: 1L9B) hã ƒe QB teƒe si wotu ene. Gake le go sia me la, RC-M polypeptide ƒe N-nuwuƒe nuto (si wɔa dɔ kple QB ƒe kadodo teƒe to Tyr ƒe nugbagbevi susɔea ƒe H kadodo si le Q helix dzi) xɔa nɔnɔme si mele dzɔdzɔme nu o, eye womegaku QB ƒe nɔnɔme ƒe tɔtrɔa me yi ŋgɔe o (30 ). Nusi na kakaɖedzi enye be míekpɔ M polypeptide ƒe tɔtrɔ sia ƒomevi le RC-LH114-W ƒe wɔwɔme me o, si sɔ kple RC-LH116 RC ƒe N-nuwuƒe nuto kloe. Ele be míade dzesii hã be le LH1 antenna si wotu ɖe atike si wotsɔna klɔa nu dzi ɖeɖeɖa vɔ megbe la, woɖe apolipoprotein RC siwo le PDB me la ɖa, si ɖe quinone-ta ememetɔwo kple ami siwo le dometsotso si le RC kple LH1 ƒe asigɛ si ƒo xlãe ƒe akpa si le eme dome la ɖa (31, 32). RC gakpɔtɔ wɔa dɔ elabena eléa cofactors katã ɖe asi, negbe QB quinone si gblẽna, si meli ke tututu o eye wòbuna zi geɖe le dzadzraɖo ƒe ɖoɖoa me koe (33). Tsɔ kpe ɖe eŋu la, wonya be LH1 kple dzɔdzɔme cyclic lipids ɖeɖeɖa tso RC me ate ŋu akpɔ ŋusẽ ɖe dɔwɔwɔwo dzi, abe P+QB-nɔnɔme si me charge-separated ƒe agbenɔƒe si dzi ɖe kpɔtɔ ene (31, 34, 35). Eyata míesusu be nutoa me LH1 ƒe asigɛ si ƒo xlã RC la ƒe anyinɔnɔ ate ŋu alé QB teƒe si “tu” la ɖe te, si ana woakpɔ nutoa me ƒe nɔnɔme si te ɖe QB ŋu ta.
Togbɔ be apolipoprotein (si me QB quinone mele o) kple eƒe wɔwɔme bliboa tsi tre ɖi na QB teƒea ƒe tɔtrɔ ƒe nɔnɔmetata eve ko hã la, ke menye nudzɔdzɔ siwo kplɔ wo nɔewo ɖo o hã la, dzesi aɖewo li be woate ŋu axe agbo na kadodoa be wòaxe mɔ ɖe gbugbɔgabla nu to hydroquinone dzi Be woaxe mɔ ɖe substrate ƒe mɔxexe nu. Quinolol kple quinone ƒe kadodo si te ɖe apolipoprotein ƒe QB teƒe ŋu ate ŋu ato vovo, si wɔnɛ be RC gbenɛ. Wogblɔ ɣeyiɣi didi aɖee nye sia be nɔnɔme ƒe tɔtrɔ wɔa akpa aɖe le quinones ƒe babla kple edzi ɖeɖe kpɔtɔ me. Ŋutete si le RC siwo wofa tsikpe me ŋu be woaɖe quinones dzi akpɔtɔ le viviti me tɔtrɔ ɖe nɔnɔmewo ŋu megbe la me gblẽ (36); X-ray crystallography ɖee fia be nusiwo gblẽ sia tso QB quinones ƒe xɔxlɔ̃ le “distal” conformation me abe 4.5 Å tso active proximal position gbɔ (26) , 37). Míeɖo aɖaŋu be didiƒe kadodo ƒe nɔnɔme sia nye domedome nɔnɔme si le apolipoprotein kple asigɛ bliboa ƒe wɔwɔme dome ƒe nɔnɔmetata, si kplɔa kadodo gbãtɔ kple quinone kple QB teƒea ƒe ʋuʋu ɖo.
RC ƒomevi II si wokpɔ le PSII complex si me fototrophic dɔlékui aɖewo kple cyanobacteria, algae kple numiemiewo le la ƒe wɔwɔme kple dɔwɔwɔ takpɔkpɔ le esi ( 38 ). Dɔwɔnu ƒe ɖoɖo si woɖe fia le nɔnɔmetata 6 (D kple E) me te gbe ɖe alesi PSII RCwo kple dɔlékui ƒe RC ƒe ƒuƒoƒoa ƒe QB teƒe ƒe sɔsɔ dzi. Ɣeyiɣi didi aɖee nye sia si wotsɔ sɔ kple wo nɔewo sia nye kpɔɖeŋu na nusɔsrɔ̃ tso quinone ƒe kadodo kple edzi ɖeɖe kpɔtɔ ƒe ɖoɖo siwo do ƒome kplikplikpli ŋu. Agbalẽ siwo wota va yi ɖee fia be nɔnɔme ƒe tɔtrɔwo kpena ɖe PSII ƒe quinones dzi ɖeɖe kpɔtɔ ŋu ( 39 , 40 ). Eyata ne míebu RC ƒe nɔnɔmetɔtrɔ ƒe takpɔkpɔ ŋu la, kadodomɔnu sia si womede dzesii tsã o ate ŋu asɔ na PSII RC ƒe QB teƒe le numiemie siwo me oxygen le siwo me keklẽŋusẽ le me.
Rps ΔpufW (pufW ɖeɖeɖa si womede dzesii o) kple PufW-His (C-nuwuƒe 10x His-tagged protein-W si woɖena fiana tso dzɔdzɔme pufW teƒe) ƒomeviwo. woƒo nu tso palustris CGA009 ŋu le míaƒe dɔ si míewɔ va yi me ( 16 ). Woɖe ƒomevi siawo kple isogenic gbemelã ƒomevi dzila tso nufamɔ̃ me to lãmenugbagbevi ʋɛ aɖewo ƒe flitete ɖe PYE (si ɖesiaɖe nye 5 g lita -1) (si wodzra ɖo ɖe LB me le -80 °C, si me 50% ( w/v) glycerol) protein, yeast extract kple succinate le) agar [1.5% (w/v)] plate dzi. Wotsɔa agbalẽvi la dea viviti me zã bliboa le xɔme ƒe dzoxɔxɔ me le nɔnɔme siwo me ya mele o me, eye emegbe wotsɔ kekeli ɣi (~50 μmolm-2 s-1) si OSRAM 116-W halogen bulbs (RS Components, UK) na la klẽe hena ŋkeke 3 va ɖo 5 vaseɖe esime lãha ɖeka nado. Wozã ƒuƒoƒo ɖeka tsɔ do M22+ medium (41) ml 10 si wotsɔ casamino acid 0.1% (w/v) kpe ɖe eŋu (si woyɔna le afisia be M22). Wodo dekɔnua le oxygen ƒe nɔnɔme si mebɔ o me le viviti me le 34°C kple ʋuʋu le 180 rpm me hena gaƒoƒo 48, eye emegbe wodo dekɔnua ƒe mililita 70 ɖe nɔnɔme ma ke me hena gaƒoƒo 24. Wozãa semi-aerobic culture si ƒe lolome nye 1 ml tsɔ doa M22 medium 30 ml ɖe 30 ml universal screw-top transparent glass bottle me eye wotsɔa ʋuʋu (~50μmolm-2 s-1) klẽna ɖe edzi gaƒoƒo 48 to magnetic force si me dɔlékui mele o Stirring rod dzi. Emegbe wodo dekɔnua ƒe mililita 30 kple dekɔnu si ade lita 1 le nɔnɔme mawo ke me, si emegbe wozã tsɔ do dekɔnu si ade lita 9 si me kekeli le ~200 μmolm-2 s-1 hena gaƒoƒo 72. Woɖe lãmenugbagbeviawo to centrifugation me le 7132 RCF me hena miniti 30, wogatsɔ wo de ~10 ml 20 mM tris-HCl (pH 8.0) me, eye wodzra wo ɖo ɖe -20°C vaseɖe esime wòhiã.
Ne èfa vɔ la, tsɔ deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) kple Roche holoenzyme protease inhibitor tablet eve (Merck, UK) ƒe kristalo aɖewo de lãmenugbagbevi siwo wogbugbɔ ƒo ɖe wo me la me. Le Franseawo ƒe nyaƒoɖeamenu ƒe lãmenugbagbevi si ƒe lolome nye 20,000 psi (Aminco, U.S.A.) me la, wogblẽ lãmenugbagbeviawo zi gbɔ zi 8 va ɖo 12. Esi woɖe lãmenugbagbevi siwo megbã o kple gbeɖuɖɔ siwo me mekɔ o ɖa to centrifugation me le 18,500 RCF me aɖabaƒoƒo 15 le 4°C me vɔ la, woɖe membrane la tso pigmented lysate me to centrifugation le 113,000 RCF me hena gaƒoƒo 2 le 43,000°C me. Tsɔ akpa si me wodea tsii la ƒu gbe eye nàgatsɔ amadede ƒe lãkusia ade 20 mM tris-HCl (pH 8.0) ml 100 va ɖo 200 me eye nàwɔ ɖeka vaseɖe esime nusiwo wotsɔ ƒo ƒui siwo woate ŋu akpɔ la nagadze o. Wotsɔ lãkusi si wotsɔ ƒo ƒui la de 20 mM tris-HCl (pH 8.0) (Anatrace, USA) si me 2% (w/v) β-DDM le me gaƒoƒo 1 le viviti me le 4°C me kple eʋuʋu blewu. Emegbe nàtsɔe aƒu gbe le dzoxɔxɔ 70°C me be wòalɔ RCF 150,000 le 4°C me gaƒoƒo 1 be nàɖe nusiwo susɔ siwo me mekɔ o ɖa.
Wotsɔ ʋuʋudedi si tso ΔpufW ƒomevi la me de 50 ml DEAE Sepharose ion exchange column si me binding buffer [20 mM tris-HCl (pH 8.0) si me 0.03% (w / v) β-DDM le ƒe sɔti ƒe lolome etɔ̃ (CV) le. Klɔ sɔti la kple CV binding buffer eve, eye emegbe nàtsɔ binding buffer eve siwo me 50 mM NaCl le klɔ sɔti la. Wotsɔ linear gradient si nye 150 va ɖo 300 mM NaCl (le binding buffer me) le 1.75 CV dzi tsi RC-LH116 complex la me, eye wotsɔ binding buffer si me 300 mM NaCl le le 0.5 CV dzi tsi binding complex susɔea. Ƒo absorption spectrum si le 250 kple 1000 nm dome, na fraction si ƒe absorbance ratio (A880/A280) lolo wu 1 le 880 va ɖo 280 nm, tsɔe ƒo zi eve le binding buffer me, eye nàgazã mɔnu ma ke le DEAE column On purification dzi. Tsɔ akpa sue siwo ƒe A880/A280 ƒe xexlẽme lolo wu 1.7 kple A880/A805 ƒe xexlẽme si kɔ wu 3.0 la ƒu gbe, wɔ ion ƒe asitɔtrɔ ƒe ƒoƒo etɔ̃lia, eye nàlé akpa sue siwo ƒe A880/A280 ƒe xexlẽme lolo wu 2.2 kple A880/A805 ƒe xexlẽme si kɔ wu 5.0 la ɖe asi. Woƒo nusi ŋu wokɔ la ƒe akpa aɖe nu ƒu va ɖo ~2 ml le Amicon 100,000 molecule weight cut-off (MWCO) centrifugal filter (Merck, UK), eye wotsɔe de Superdex 200 16/600 size exclusion column (GE Healthcare, US) si me 200 mM NaCl buffer le dzi, eye emegbe woɖee le buffer ma ke me le 1.5 CV me. Ƒo absorption spectra siwo le lolome ƒe ɖeɖeɖa ƒe akpa la nu ƒu, eye nàƒo absorption spectra siwo ƒe A880/A280 ƒe xexlẽme le 2.4 kple A880/A805 ƒe xexlẽme si wu 5.8 va ɖo 100 A880 nu ƒu, eye nàzã wo enumake hena cryo-TEM grid dzadzraɖo alo edzadzraɖo Na wodzra wo ɖo ɖe -80°C vaseɖe esime wòahiã.
Wotsɔ ʋuʋudedi si tso PufW-His ƒomevi me la de 20 ml HisPrep FF Ni-NTA Sepharose kɔlam (20 mM tris-HCl (pH 8.0) si me 200 mM NaCl kple 0.03% (w/w) le) le IMAC buffer ( GE Healthcare) me. v) β-DDM ƒe dɔwɔwɔ]. Wotsɔ IMAC buffer ƒe CV atɔ̃ klɔ sɔti la, eye emegbe wotsɔ IMAC buffer ƒe CV atɔ̃ siwo me histidine 10 mM le. Wotsɔ IMAC buffer atɔ̃ siwo me histidine 100 mM le la ɖe core complex la ɖa le sɔti la me. Woƒoa akpa si me RC-LH114-W ƒe ƒuƒoƒoa le la nu ƒu wòdea ~10 ml le tsigoe si woʋuʋu si me Amicon 100,000 MWCO filter (Merck, UK) le me, wotsɔa binding buffer tsia eme zi 20, eye emegbe wotsɔa binding buffer dea eme zi 25 ml Le DEAE Sepharose column me la, wozãa CV ene siwo bla ɖe buffer la ŋu do ŋgɔ. Klɔ sɔti la kple CV binding buffer ene, emegbe nàkɔ complex la le CV enyi dzi le linear gradient si nye 0 va ɖo 100 mM NaCl (le binding buffer me), kple CV ene susɔe siwo me 100 mM binding buffer le The residual complexes eluted on the sodium chloride combined with the A880/A280 ratio higher than 2.4 and the Woƒo A880/A805 ƒe xexlẽme si kɔ wu 4.6 ƒe akpa suewo nu ƒu va ɖo ~2 ml le Amicon 100,000 MWCO centrifugal filter me, eye wotsɔ 1.5 CV IMAC yɔe do ŋgɔ Buffer equilibrated Superdex 200 16/600 size exclusion column, eye emegbe woɖee le buffer ma ke me le 1.5 CV dzi. Ƒo absorption spectra siwo le size-exclusion fractions me nu ƒu eye nàƒo absorption spectrawo nu ƒu kple A880/A280 ratios si wu 2.1 kple A880/A805 ratios si wu 4.6 va ɖo 100 A880, siwo wozãna enumake hena TEM grid si wofa tsikpe me dzadzraɖo alo wodzraa wo ɖo ɖe -80°C vaseɖe esime wòhiã.
Wozã Leica EM GP immersion freezer tsɔ dzra TEM grids siwo me dzoxɔxɔ le bɔbɔe ɖo. Wotsɔ IMAC buffer ƒo complex la ɖe A880 si nye 50 me, eye emegbe wotsɔ 5μl de QUANTIFOIL 1.2/1.3 carbon-coated copper mesh (Agar Scientific, UK) si me keklẽ yeye le si woɖe tso eme la dzi. Tsɔ grid la de 20°C kple 60% relative humidity me hena s 30, emegbe nàtutui wòaƒu hena s 3, eye emegbe nàtsie le ethane si nye tsi me le -176°C.
Woŋlɔ RC-LH114-W complex ƒe nyatakakawo ɖe eBIC (Electronic Bioimaging Center) (British Diamond Light Source) dzi kple Titan Krios nusuekpɔmɔ̃, si wɔa dɔ le elektrikŋusẽ si le du dzi si nye 300kV, si ƒe lolome le ŋkɔ me nye 130,000× kple ŋusẽ si nye- Tia dometsotso si nye 20 eV. Wozã Gatan 968 GIF Quantum si me K2 peak detector le tsɔ ŋlɔ nɔnɔmetatawo le xexlẽdzesiwo ƒe nɔnɔme me tsɔ ƒo nyatakakawo nu ƒu. Pixel ƒe lolome si wodo kpɔ nye 1.048Å, eye agbɔsɔsɔme si wozãnae nye 3.83 e-Å-2s-1. Woƒo sinima la nu ƒu le sɛkɛnd 11 me eye womae ɖe akpa 40 me. Zã teƒe si wotsɔ carbon ƒo xlãe nàtsɔ agbugbɔ ŋku alé ɖe nusuekpɔmɔ̃a ŋu, eye emegbe nàƒo sinima etɔ̃ nu ƒu ɖe do ɖesiaɖe me. Le wo katã me la, woƒo sinima 3130 nu ƒu, eye woƒe defocus ƒe asixɔxɔwo le -1 kple -3μm dome.
Wozã nusuekpɔmɔ̃ ma ke tsɔ ƒo nyatakakawo nu ƒu tso RC-LH116 ƒe ƒuƒoƒoa ŋu le Asterbury Biostructure Laboratory (University of Leeds, UK). Woƒo nyatakakaawo nu ƒu le xexlẽme ƒe nɔnɔme me kple dzidziɖedzi 130 k, eye wodo piksel ƒe lolome ɖe 1.065 Å kple agbɔsɔsɔme si nye 4.6 e-Å-2s-1. Wolé sinima la ɖe mɔ̃ dzi le sɛkɛnd 12 me eye womae ɖe akpa 48 me. Le wo katã me la, woƒo film 3359 nu ƒu, eye defocus ƒe asixɔxɔwo le -1 kple -3μm dome.
Wowɔa nyatakakawo ŋuti dɔwɔwɔ katã le Relion 3.0 ƒe mɔ̃a me (42). Zã Motioncorr 2 (43) nàtsɔ aɖɔ beam ƒe ʋuʋu ɖo to agbɔsɔsɔme ƒe kpekpeme dzi, eye emegbe nàzã CTFFIND 4.1 (44) atsɔ anya CTF (contrast transfer function) parameter. Foto suesuesue siwo bɔ le dɔwɔwɔ ƒe afɔɖeɖe gbãtɔ siawo megbe la dze le Nɔnɔmetata 2. S16. Wowɔa tiatiawɔwɔ ƒe nɔnɔmetata si wowɔna le wo ɖokui si to asi tiatia si ade piksel 250 siwo me nusuekpɔmɔ̃ 1000 le le piksel 250 ƒe xɔtunu me eye womekpɔa nufiame eve ƒe hatsotso (2D) ƒe hatsotso aɖeke o, si wɔnɛ be wogbea hatsotso mawo siwo doa go kpɔɖeŋu ƒe ɖiƒoƒo alo nɔnɔme aɖeke si woate ŋu ade dzesii o. Emegbe wowɔ tiatia le wo ɖokui si le foto sueawo katã dzi, eye RC-LH114-W nye nusuekpɔmɔ̃ 849,359, eye RC-LH116 ƒe ƒuƒoƒoa nye nusuekpɔmɔ̃ 476,547. Wogbea nusue siwo katã wotia la to 2D ƒe hatsotso si menye nufiame o ƒe ƒoƒo eve me, eye le duƒuƒu ɖesiaɖe megbe la, wogbea nusue siwo do go carbon ƒe teƒea, kpɔɖeŋu ƒe ɖiƒoƒo, nɔnɔme aɖeke si dze ƒã alo nusue siwo ƒo xlã wo nɔewo sesĩe o, si wɔe be 772,033 (90.9%) kple 359,678 (75.5%) ) Wozãa nusuewo hena 3D hatsotso ƒe RC-LH114-W kple RC-LH116 le wo nɔewo yome. Wozã stochastic gradient descent mɔnu tsɔ wɔ 3D ƒe nufiame kpɔɖeŋu gbãtɔ. Wozã kpɔɖeŋu gbãtɔa abe nusi dzi woanɔ te ɖo ene la, woma nusuekpɔmɔ̃ siwo wotia la ɖe hatsotso ene me le 3D me. Zã kpɔɖeŋu si le hatsotso sia me abe nufiame ene, wɔ 3D kɔklɔ ɖe nusue siwo le hatsotso gãtɔ me dzi, emegbe nàzã 15Å low-pass filter gbãtɔa atsɔ atsyɔ solvent ƒe teƒea dzi, atsɔ piksel 6 ƒe nugbɔ falɛfalɛwo akpe ɖe eŋu, eye nàtrɔ asi le pikselawo ŋu le emegbe atsɔ aɖɔ Gatan K2 peak Modulation transfer function si le top detector la ɖo. Le RC-LH114-W nyatakakadzraɖoƒe la, wotrɔ asi le kpɔɖeŋu gbãtɔ sia ŋu to ʋuʋu sesẽ si le mask la ƒe goawo dzi (si woɖe tso core complex density le UCSF Chimera me) ɖeɖeɖa me. Wozãa kpɔɖeŋu siwo do tso eme (RC-LH114-W kple RC-LH116 ƒe vovototodedeameme nye 3.91 kple 4.16 Å, le wo nɔewo yome) abe nusi woatsɔ akpɔ 3D ƒe hatsotso evelia me ene. Woƒo nusuekpɔmɔ̃ siwo wozã la nu ƒu ɖe 3D ƒe hatsotso gbãtɔ me eye kadodo sẽŋu aɖeke mele wo kple nutoa me o. Xɔtuɖoɖo ƒe nɔnɔme siwo dze ƒã ƒe ƒoƒo ɖe wo nɔewo dzi alo woƒe anyimanɔmanɔ. Le 3D ƒe hatsotso evelia megbe la, wotia hatsotso si ƒe lolome lolo wu [Le RC-LH114-W gome la, hatsotso ɖeka nye nusuekpɔmɔ̃ 377,703 (44.5%), le RC-LH116 gome la, hatsotso eve li, siwo katã nye nusue 260,752 (54.7%) , Afisi wosɔ le ne woɖo wo ɖe ɖoɖo nu le tɔtrɔ gbãtɔ megbe ko kple vovototo sue aɖe]. Wogaɖea nu suesuesue siwo wotia la le aɖaka si ƒe lolome nye piksel 400 me eye wotoa 3D ƒe asitɔtrɔ le wo ŋu me. Wowɔa solvent mask la to 15Å low-pass filter gbãtɔ, 3 pixel map kekeɖenu kple 3 pixel soft mask zazã me. Wozãa nusue ɖesiaɖe ƒe CTF ƒe asitɔtrɔ, nusue ɖesiaɖe ƒe ʋuʋu ɖɔɖɔɖo kple nusue ɖesiaɖe ƒe CTF ɖɔɖɔɖo ƒe ƒoƒo evelia, wowɔa 3D ɖɔɖɔɖo, solvent masking kple post-processing le afɔɖeɖe ɖesiaɖe megbe be woagaɖɔ nɔnɔme si do tso eme la ɖo. Ne míezã FSC (Fourier Shell Correlation Coefficient) ƒe cut-off value si nye 0.143 la, RC-LH114-W kple RC-LH116 ƒe kpɔɖeŋu mamlɛawo ƒe nyametsotsowo nye 2.65 kple 2.80Å, le wo nɔewo yome. FSC curve si le model mamlɛtɔ me la dze le Figure 2. S17.
Woɖea protein ƒe ɖoɖowo katã tso UniProtKB: LH1-β (PufB; UniProt ID: Q6N9L5); LH1-α (PufA; UniProtID: Q6N9L4); RC-L (PufL; UniProt ƒe xexlẽdzesi: O83005); RC-M (PufM; UniProt ƒe ID: A0A4Z7); RC-H (PuhA; UniProt ƒe xexlẽdzesi: A0A4Z9); Protein-W (PufW; UniProt ƒe xexlẽdzesi: Q6N1K3). Wozã SWISS-MODEL (45) tsɔ wɔ RC ƒe homology model, si me protein ƒe ɖoɖo siwo le RC-L, RC-M kple RC-H kple Rba ƒe kristalo wɔwɔme le. wozã sphaeroides RC abe nɔnɔmetata ene (PDB ID: 5LSE) (46). Zã dɔwɔnu si nye “fit map” le UCSF Chimera me nàtsɔ awɔ kpɔɖeŋu si wowɔ la wòasɔ ɖe anyigbatataa nu (47), nàna protein ƒe wɔwɔme nanyo ɖe edzi, eye nàtsɔ cofactor [4×BChl a (monomer agbalẽdzraɖoƒe ƒe nugbagbevi susɔe ƒe ŋkɔ = BCL), 2×BPh a (BPH), UQ10 ƒomevi ɖeka alo eve (U10), non-heme iron (Fe) ɖeka kple 3,4-dihydrohexacarbonylcholine ɖeka (QAK)] zã Coot (48) tsɔ kpe ɖe eŋu. Esi wònye be QAK mele monomer agbalẽdzraɖoƒea o ta la, wotsɔ eLBOW dɔwɔnu si le PHENIX (49) me tsɔ parameter nae.
Eyome wowɔ LH1 ƒe akpa suea. Le gɔmedzedzea me la, wozã xɔtudɔwɔnu si le eɖokui si le PHENIX (49) me tsɔ tu LH1 ƒe ɖoɖoa ƒe akpa aɖe le eɖokui si tsɔ zã anyigbatata la kple LH1-α kple LH1-β protein ƒe ɖoɖo siwo wotsɔ dea eme. Tia LH1 ƒe akpa sue si de blibo wu, ɖee eye nàtsɔe ade Coot me, atsɔ asi atsɔ ɖoɖo si bu la akpe ɖe eŋu, eye nàtsɔ asi atrɔ asi le wɔwɔme bliboa ŋu hafi nàtsɔ BCl eve a (BCL) kple spirilloxanthin (CRT) akpe ɖe eŋu [le Rps si sɔ nu LH1 complex ƒe kpekpeme kple carotenoid ƒe agbɔsɔsɔ si wonya. Lã ƒomeviwo (17)]. Kɔpi LH1 ƒe akpa sue bliboa, eye nàzã UCSF Chimera “Docking Map Tool” atsɔ atɔ ɖe teƒe si te ɖe eŋu si menye kpɔɖeŋu o si me LH1 ƒe kpekpeme le, eye emegbe nàtrɔ asi le eŋu le Coot me; gbugbɔ wɔ dɔa vaseɖe esime woawɔ LH1 ƒe akpa suewo katã ƒe kpɔɖeŋu. Le RC-LH114-W ƒe wɔwɔme gome la, to ʋeʋẽ si womeɖo ɖe Coot me o ɖeɖe me la, woma protein la tso akpa susɔe siwo menye protein o le USCF Chimera ƒe anyigbatata me eye wozãa Autobuild dɔwɔnu tsɔ ɖoa ​​kpɔɖeŋu gbãtɔ, kple subunit susɔeawo (protein-W) Modeling. Le PHENIX (49) me. Tsɔ tsiatsia ɖesiaɖe si bu kpe ɖe kpɔɖeŋu si do tso eme le Coot (48) me ŋu, eye emegbe nàtsɔ asi atrɔ asi le subunit bliboa ŋu. Density susɔe si womeɖo o la sɔ kple lipids (PDB monomer agbalẽdzraɖoƒe ID si nye CDL = CDL, POPC = 6PL kple POPG = PGT), β-DDM detergent (LMT) kple UQ10 molecules (U10) ƒe ƒuƒoƒo. Zã PHENIX ƒe nyonyome (49) kple asi ƒe nyonyome le Coot (48) me nàtsɔ ade blibo gbãtɔ ƒe kpɔɖeŋu bliboa vaseɖe esime womate ŋu awɔ kpɔɖeŋua ƒe akɔntabubuwo kple nukpɔkpɔ ƒe nyonyome si sɔ ɖe enu wòanyo ɖe edzi o. Mlɔeba la, zã LocScale (50) nàtsɔ aƒo nutoa me ƒe anyigbatata la nu ƒu, eye emegbe nàwɔ tsatsam bubu geɖe siwo nye modeling the unallcated density kple automatic and manual optimization.
Peptide, cofactors kple lipid kple quinones bubu siwo docked le woƒe densities me la dze le Nɔnɔmetata 1 kple 2. S18 vaseɖe S23. Woɖe akɔntabubu ŋuti nyatakaka siwo le kpɔɖeŋu mamlɛtɔa ŋu fia le Tabla S1 me.
Negbe ɖe wogblɔe bubui ko la, woƒo UV/Vis/NIR ƒe absorption spectra nu ƒu ɖe Cary60 spectrophotometer (Agilent, USA) dzi le 1 nm dometsotsowo me tso 250 nm va ɖo 1000 nm kple ƒoƒo ɖekae ƒe ɣeyiɣi si nye 0.1s.
Tsɔ kpɔɖeŋua ƒo ɖe quartz cuvette si ƒe mɔ nye mm 2 yi A880 si nye 1 me, eye nàƒo absorption spectrum si le 400 kple 1000 nm dome nu ƒu. Woƒo dichroic spectra siwo le gota la nu ƒu ɖe Jasco 810 spectropolarimeter (Jasco, Japan) dzi le 1 nm dometsotso siwo le 400 nm kple 950 nm dome le scan rate si nye 20 nm min-1 me.
Wokpɔa molar ƒe tsɔtsrɔ̃ ƒe xexlẽdzesi to core complex la ɖeɖeɖa ɖe A880 si ade 50. Tsɔ 10μl ƒe agbɔsɔsɔme si le 990μl binding buffer alo methanol me, eye nàƒo absorption spectrum la nu ƒu enumake be BChl ƒe gbegblẽ dzi naɖe akpɔtɔ. Wobu BChl ƒe agbɔsɔsɔ le methanol ƒe kpɔɖeŋu ɖesiaɖe me to tsɔtsrɔ̃ ƒe xexlẽdzesi si le 771 nm si nye 54.8 mM-1 cm-1, eye wokpɔ tsɔtsrɔ̃ ƒe xexlẽdzesi ( 51 ). Ma BChl ƒe agbɔsɔsɔ si wodzidze la ɖe 32 (RC-LH114-W) alo 36 (RC-LH116) me be nànya core complex ƒe agbɔsɔsɔme, si emegbe wozãna tsɔ nyaa absorption spectrum si le kpɔɖeŋu ma ke si woƒo ƒu le buffer Extinction coefficient me. si le sɔsɔnu. Wodzidze nu etɔ̃ enuenu na kpɔɖeŋu ɖesiaɖe, eye wozã BChl Qy ƒe agbɔsɔsɔ si sɔ gbɔ wu ƒe absorbance le mama dedie nu tsɔ bu akɔnta. RC-LH114-W ƒe tsɔtsrɔ̃ ƒe xexlẽdzesi si wodzidze le 878 nm nye 3280±140 mM-1 cm-1, gake RC-LH116 ƒe tsɔtsrɔ̃ ƒe xexlẽdzesi si wodzidze le 880 nm nye 3800±30 mM-1 cm-1.
Wobu UQ10 ƒe agbɔsɔsɔme le mɔnu si le (52) me nu. Kpuie ko la, wozã Agilent 1200 HPLC ɖoɖoa tsɔ wɔ reverse phase HPLC (RP-HPLC). Tsɔ RC-LH116 alo RC-LH114-W si ade 0.02 nmol ƒo ɖe 50μl methanol:chloroform 50:50 si me ferric chloride 0.02% (w/v) le me, eye nàdo Beckman Coulter Ultrasphere ODS 4.6 mm si woda sɔ do ŋgɔ la ɖe eme atike (80:20 methanol:2-propanol) le sɔti si nye ×25 cm dzi. Wɔ isocratic elution le HPLC solvent me be nàlé ŋku ɖe absorbance ŋu le 275 nm (UQ10), 450 nm (carotenoids) kple 780 nm (BChl) hena gaƒoƒo 1. Wotsɔ taƒoƒo si le 275 nm chromatogram me le miniti 25.5 me la ƒo ƒu, si me atike bubu aɖeke si woate ŋu ade dzesii mele o. Wozãa teƒe si wotsɔ ƒo ƒui tsɔ bua UQ10 ƒe molar agbɔsɔsɔme si woɖe tso calibration curve si wobu tso dzidzenu dzadzɛwo dodo tso 0 va ɖo 5.8 nmol (Nɔnɔmetata S14). Woda kpɔɖeŋu ɖesiaɖe kpɔ le zi etɔ̃ me, eye vodada si ŋu woka nya ta le la sɔ kple SD si le mamã dedie nu.
Wowɔ tsi si me RC-LH1 ƒe ƒuƒoƒoa le si ƒe Qy ƒe xɔxlɔ̃ si sɔ gbɔ wu 0.1 kple sɔ ƒe dzi ƒe cytochrome c2 si dzi ɖe kpɔtɔ 30 μM (Merck, UK) kple 0 va ɖo 50 μMUQ2 (Merck, UK). Wodzra 1-ml ƒe kpɔɖeŋu etɔ̃ ɖo le UQ2 ƒe agbɔsɔsɔ ɖesiaɖe me eye wotsɔ wo de viviti me zã bliboa le 4°C be woakpɔ egbɔ be wotrɔ ɖe viviti ŋu bliboe hafi wodzidze wo. Wotsɔ atike la de OLIS RSM1000 modular spectrophotometer si me wotsɔ 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm titina kple 0.6 mm outlet slits ɖo. Wotsɔa 600 nm didi pass filter dana ɖe kpɔɖeŋu fototube kple nufiame fotomultiplier tube ƒe mɔnu be woaɖe dzidzɔdonamekekeli ɖa. Wolé ŋku ɖe absorbance ŋu le 550 nm kple integration time si nye 0.15 s. Wodoa dzidzɔkpɔkpɔ ƒe kekeli tso 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) to fiber optic ka dzi le 90% ƒe sesẽme to DC2200 dziɖula (Thorlabs Ltd., UK) dzi eye wòdona ɖe kekelitsoƒea le dzogoe si nye 90° ƒe. Wotsɔa dzidzenua tsi tre ɖe ahuhɔ̃ea ŋu be wòatrɔ kekeli ɖesiaɖe si kpɔɖeŋua mexɔ le gɔmedzedzea me o la atrɔ. Kpɔ absorbance s 10 hafi kekeli 50 s. Emegbe wogalé ŋku ɖe absorbance ŋu hena s 60 le viviti me be woakpɔ alesi gbegbe quinolol ɖea cytochrome c23 + dzi kpɔtɔna le eɖokui si (kpɔ Nɔnɔmetata S8 hena nyatakaka xoxowo).
Wowɔ dɔ tso nyatakakaawo ŋu to flidzidzedze ƒe tsɔtsɔme si sɔ ɖe ɖoɖo nu le s 0.5 va ɖo 10 me (le UQ2 ƒe agbɔsɔsɔme nu) kple kpɔɖeŋu etɔ̃awo katã ƒe agbɔsɔsɔme ƒe mamã dedie le UQ2 ƒe agbɔsɔsɔme ɖesiaɖe me. Wozã RC-LH1 ƒe agbɔsɔsɔ si wobu to tsɔtsrɔ̃ ƒe xexlẽdzesi ɖesiaɖe dzi tsɔ trɔ agbɔsɔsɔmea ɖe nusiwo naa nu ʋãna ƒe dɔwɔwɔ nyuie me, wowɔe ɖe Origin Pro 2019 (OriginLab, USA) me, eye wotsɔe sɔ kple Michaelis-Menten ƒe kpɔɖeŋua tsɔ de dzesi Km kple Kcat ƒe asixɔxɔ siwo dze abe ɖe wòdze ene.
Le ɣeyiɣi kpui aɖe ƒe absorption dzidzedzewo gome la, woɖe RC-LH1 ƒe kpɔɖeŋua ɖe ~2μM le IMAC buffer si me 50 mM sodium ascorbate (Merck, USA) kple 0.4 mM Terbutin (Merck, USA) le me. Wozãa ascorbic acid abe vɔsa elektrɔnik nunala ene, eye wozãa tert-butaclofen abe QB mɔxenu ene tsɔ kpɔa egbɔ be RC nunala vevitɔa gakpɔtɔ dzi ɖe kpɔtɔ (si fia be menye fotooxidized o) le dzidzedze ƒe ɖoɖo bliboa me. Wotsɔa kpɔɖeŋu si ade mililita 3 dea lãmenugbagbevi si trɔna si wowɔ ɖe ɖoɖo nu (si ƒe didime anɔ abe 0.1 m ene, 350 RPM) si ƒe didime nye milimeta 2 le nukpɔkpɔmɔa me be woakpɔ egbɔ be ɣeyiɣi si sɔ na kpɔɖeŋu si le laser mɔa me na viviti me tɔtrɔ le dzidzɔdoname ƒe ʋuʋudediwo dome. Zã ~100-fs laser pulses tsɔ do ŋusẽ Ti: Sapphire laser system (Spectra Physics, USA) tsɔ do dzidzɔ na kpɔɖeŋua le 880 nm le gbugbɔgagblɔ ƒe tsɔtsɔme si nye 1 kHz (20 nJ na NIR alo 100 nJ na Vis). Hafi nàƒo nyatakakawo nu ƒu la, tsɔ kpɔɖeŋua de kekeli si doa dzidzɔ na ame me abe miniti 30 ene. Ne wodoe ɖe go ana QA nagawɔ dɔ o (ɖewohĩ ana QA dzi naɖe akpɔtɔ zi ɖeka alo zi eve). Gake taflatse de dzesii be woate ŋu atrɔ ɖoɖo sia elabena le viviti me tɔtrɔ ɖe nɔnɔme ŋu ɣeyiɣi didi aɖe megbe la, RC atrɔ ayi QA dɔwɔna me vivivi. Wozã Helios spectrometer (Ultrafast Systems, U.S.A.) tsɔ dzidze ɣeyiɣi kpui aɖe ƒe ɣeyiɣi ƒe didime kple ɣeyiɣi si wohe ɖe megbe si nye -10 va ɖo 7000 ps . Zã Surface Xplorer kɔmpiuta dɔwɔɖoɖo (Ultrafast Systems, U.S.A.) nàtsɔ aɖe nyatakakaawo ƒe hatsotsowo ɖa, emegbe nàtsɔ wo aƒo ƒui ahawɔ wo ɖe ɖoɖo nu. Zã CarpetView kɔmpiuta dɔwɔɖoɖo (Light Conversion Ltd., Lithuania) nàtsɔ azã nyatakaka siwo woƒo ƒu atsɔ akpɔ vovototo ƒe spektrum siwo ku ɖe gbegblẽ ŋu, alo zã dɔwɔwɔ si ƒoa xexlẽdzesi geɖe kple dɔwɔnu ƒe ŋuɖoɖo be wòasɔ ɖe ƒutsotsoe ƒe didime ɖeka ƒe spektral ƒe tɔtrɔ le Origin (OriginLab, USA) me.
Abe alesi míegblɔe le etame ene (53) la, wodzra keklẽŋusẽ ƒe film si me LH1 complex si me RC kple peripheral LH2 antenna siaa mele o ɖo. Wotsɔ 20 mM tris (pH 8.0) ƒo lãkusia eye emegbe wotsɔe de quartz cuvette si me 2 mm ƒe nukpɔkpɔmɔ le me. Wozã 30nJ laser pulse tsɔ do dzidzɔ na kpɔɖeŋua le 540 nm kple megbedede ƒe ɣeyiɣi si nye -10 va ɖo 7000 ps. Wɔ dɔ tso nyatakakawo ƒe ƒuƒoƒoa ŋu abe alesi woɖe eme na Rps. pal ƒe kpɔɖeŋu.
Wotsɔ centrifugation wɔ membrane la le centrifugation le 150,000 RCF me hena gaƒoƒo 2 le 4°C, eye emegbe wogbugbɔ tsɔ eƒe absorbance le 880 nm de 20 mM tris-HCl (pH 8.0) kple 200 mM NaCl me. Tsɔ lãkusia ƒo ɖe 2% (w/v) β-DDM me blewuu gaƒoƒo 1 le viviti me le 4°C me. Wotsɔ 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) ƒo kpɔɖeŋua ɖe protein ƒe agbɔsɔsɔ si nye 2.5 mg ml-1 (Bio-Rad numekuku). Wogawɔ dɔ bubuwo tso mɔnu si wota va yi (54) me, wodze egɔme kple protein 50 μg ƒe tsinyenye ɖe TEAB 50 μl katã si me sodium laurate 1% (w/v) le (Merck, UK) me. Le sonication na 60 s megbe la, woɖee dzi kpɔtɔ kple 5 mM tris (2-carboxyethyl) phosphine (Merck, UK) le 37 ° C hena miniti 30. Le S-alkylation gome la, tsɔ 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) de kpɔɖeŋua me eye nàtsɔe akpe ɖe eŋu tso 200 mM isopropanol stock solution me aɖabaƒoƒo 10 le xɔme ƒe dzoxɔxɔ nu. Wowɔ proteolytic nuɖuɖumeŋusẽ to trypsin/endoproteinase Lys-C tsakatsaka (Promega UK) 2 μg dede eme eye wotsɔe de dzoxɔxɔ 37°C me gaƒoƒo 3. Woɖe laurate surfactant la to 50 μl ethyl acetate kple 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) dede eme kple vortexing hena s 60. Wodo phase ƒe mama ɖe ŋgɔ to centrifugation le 15,700 RCF me hena miniti 5. Le adzɔnuwɔƒea ƒe ɖoɖo nu la, wozã C18 spin column (Thermo Fisher Scientific, UK) tsɔ ƒo peptide la ƒe akpa si le ete la nyuie heɖe dze le eme. Le ƒuƒu to vacuum centrifugation megbe la, wotsɔ kpɔɖeŋua ƒo ɖe 0.5% TFA kple 3% acetonitrile me, eye wotsɔ nanoflow RP chromatography kpe ɖe mass spectrometry ŋu da 500 ng kpɔ to ɖoɖoa ƒe nɔnɔme siwo ŋu míeƒo nu tsoe tsitotsito va yi zazã me.
Zã MaxQuant v.1.5.3.30 (56) hena protein ƒe dzesidede kple eƒe agbɔsɔsɔme be nàdi Rps. palustris proteome ƒe nyatakakadzraɖoƒe (www.uniprot.org/proteomes/UP000001426). Wotsɔ mass spectrometry proteomics nyatakakawo da ɖe ProteomeXchange Alliance to PRIDE partner nudzraɖoƒe (http://proteomecentral.proteomexchange.org) le dataset identifier PXD020402 te.
Le numekuku to RPLC me tsɔ kpe ɖe electrospray ionization mass spectrometry ŋu la, wodzra RC-LH1 ƒe ƒuƒoƒoa ɖo tso gbemelã ƒomevi Rps me. Ne wozã mɔnu si wota va yi (16) la, protein ƒe agbɔsɔsɔ si wowɔ le palustris lãmenugbagbeviwo me nye 2 mg ml-1 le 20 mM Hepes (pH 7.8), 100 mM NaCl kple 0.03% (w/v) β- (Bio-Rad numekuku) ) DDM me. Le amesi wɔe ƒe ɖoɖo nu la, zã 2D purification kit (GE Healthcare, USA) nàtsɔ aɖe protein 10 μg to tsidzadza mɔnu dzi, eye nàtsɔ tsidzadzaa alɔ 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile kple 20% (v/v) tsi me. Wotsɔ RPLC (Dionex RSLC) si wotsɔ kpe ɖe mass spectrometry (Maxis UHR-TOF, Bruker) ŋu da microliter atɔ̃ kpɔ. Zã MabPac 1.2×100 mm sɔti (Thermo Fisher Scientific, UK) hena mama le 60°C kple 100μlmin -1, kple 85% (v / v) ƒe ʋeʋẽ A [0.1% (v / v) FA kple 0.02% (V/v) TFA tsi ƒe ʋeʋẽ] va ɖo 85%(v/v) ʋeʋẽ B [0.1%(v/v) FA kple 0.02%(v/v) le 90%(v/v) acetonitrile TFA me] Ne wozã electrospray ionization dzɔtsoƒe si wozãna ɖaa kple default parameters hena wu miniti 60 la, mass spectrometer la xɔa 100 va ɖo 2750 m/z (mass-to-charge ratio). To ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/) ƒe kpekpeɖeŋu me la, wɔ nɔnɔmetata na mass spectrum la ɖe subunits si le complex la me.
Wodzi lãmenugbagbeviawo gaƒoƒo 72 le 100 ml NF-low (10μMm-2 s-1), medium (30μMm-2 s-1) alo high (300μMm-2 s-1) kekeli te. M22 medium (M22 medium si me woɖea ammonium sulfate ɖa le eye wotsɔa sodium acetate ɖɔlia sodium succinate) le 100 ml ƒe aŋetu si wotsɔ gaviwo wɔe me (23). Le s 30 ƒe tsatsam atɔ̃ me la, wotsɔa 0.1 micron glass beads ƒo ƒu le woƒe lolome ƒe sɔsɔme 1:1 me be woaɖe lãmenugbagbeviawo ɖa eye wofa wo ɖe tsikpe dzi aɖabaƒoƒo 5. Woɖe nusi me mekɔ o, lãmenugbagbevi siwo megbã o kple glass beads ɖa to centrifugation le 16,000 RCF me hena miniti 10 le benchtop microcentrifuge me. Woɖe lãkusia ɖe vovo le Ti 70.1 rotor si me RCF 100,000 le le 20 mM tris-HCl (pH 8.0) me kple 40/15% (w/w) sucrose gradient me hena gaƒoƒo 10.
Abe alesi míegblɔe le míaƒe dɔ si míewɔ va yi me ene la, immunodetection of the His tag on PufW (16). Kpuie ko la, core complex si ŋu wokɔ (11.8 nM) alo membrane si me RC ƒe agbɔsɔsɔ ɖeka le (si wokpɔna to oxidation si woɖe vovototo ƒe spektrum si dzi ɖe kpɔtɔ kple agba si le stained gel dzi sɔsɔ me) le 2x SDS loading buffer (Merck, UK) me si woɖe zi eve. Woɖe proteinawo ɖe vovo le 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK) si wogbugbɔ wɔ dzi. Wotsɔ Coomassie Brilliant Blue (Bio-Rad, UK) de ama gel aɖe be woatsɔ akɔ RC-L ƒe akpa suea ahakpɔe le susu me. Wotsɔ protein si le gel evelia dzi la de methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) me hena dɔlélenutsiŋutete dodokpɔ. Woxe mɔ ɖe PVDF ƒe lãkusia nu le 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 kple 5% (w / v) notsi babla si me wode tsii me, eye emegbe wotsɔe kple anti-His primary antibody (le Dilute the antibody buffer [50 mM tris-HCl (pH 7.6) me), tsɔe de lãme na ame. 150 mM NaCl kple 0.05% (v/v) Tween-20] le 1:1000 A190-114A, Bethyl Laboratories, U.S.A.) me hena gaƒoƒo 4. Esi woklɔ lãkusia zi 3 hena aɖabaƒoƒo 5 le dɔlékuiwutikewo ƒe buffer me vɔ la, wotsɔ horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (si woɖe 1:10,000 le antibody buffer me) ƒo lãkusia nu ƒu be wòana woate ŋu ade dzesii (aɖabaƒoƒo 5 le tsilele zi 3 le antibody buffer me megbe) to WESTAR ETA C 2.0 chemiluminescence substrate zazã me (Cyanagen, Italy) kple Amersham Imager 600 (GE Healthcare, UK).
To gel alo dɔlélenutsiŋutete dodokpɔ ƒe mɔ ɖesiaɖe ƒe sesẽme mama tata, nuto si le taƒoƒoa te tsɔtsɔ ƒo ƒui kple akɔntabubu le RC-L (gel si ŋu wode amae) kple Protein-W (dɔlélenutsiŋutete dodokpɔ) ƒe sesẽme ƒe sɔsɔme ŋu, le ImageJ (57) Dɔwɔwɔ ɖe nɔnɔmetata la ŋu me. Wotrɔ xexlẽme siawo wozu molar ƒe xexlẽme to ebubu be RC-L kple protein-W ƒe xexlẽme le RC-LH114-W ƒe kpɔɖeŋu dzadzɛa me nye 1:1 eye wowɔ nyatakaka bliboa wòsɔ ɖe ɖoɖo nu.
Ne èdi kpeɖeŋutɔ nyati sia la, taflatse kpɔ http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
Esia nye nyati si woate ŋu akpɔ le mɔ gbadza nu si woma le Creative Commons Attribution License ƒe sewo nu. Nyatia ɖe mɔ ɖe eŋudɔwɔwɔ, emama, kple egbugbɔgata mɔxeɖenu aɖeke manɔmee le mɔnu ɖesiaɖe me nenye be woyɔ dɔ gbãtɔa nyuie.
De dzesii: Ðeko míebia tso asiwò be nàtsɔ wò e-mail adrɛs ana ale be amesi nèkafu na axaa nanya be yedi be yeakpɔ e-mail la eye be menye nyatakaka gbegblẽe wònye o. Mímalé e-mail adrɛs aɖeke ɖe mɔ̃ dzi o.
Wozãa biabia sia tsɔ doa amedzro kpɔ nenye be amedzroe nènye eye woxea mɔ na nyatakaka gbegblẽwo ɖoɖo ɖe amewo le wo ɖokui si.
David J. K. Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Agbledela, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P. Canniffe , Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Kekeli ƒe mɔ̃ 1 ƒe wɔwɔme si ƒe nɔnɔme deŋgɔ le nuwɔna ƒe teƒea naa gɔmesese yeyewo le quinone ƒe ŋusẽkpɔɖeamedzi ŋu.
David J. K. Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Agbledela, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P. Canniffe , Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Kekeli ƒe mɔ̃ 1 ƒe wɔwɔme si ƒe nɔnɔme deŋgɔ le nuwɔna ƒe teƒea naa gɔmesese yeyewo le quinone ƒe ŋusẽkpɔɖeamedzi ŋu.
©2021 Amerikatɔwo ƒe Dzɔdzɔmeŋutinunya ƒe Ŋgɔyiyi Habɔbɔ. gomenɔamesiwo katã le esi. AAAS nye HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef kple COUNTER ƒe hadɔwɔla. Dzɔdzɔmeŋutinunya ƒe Ŋgɔyiyi ISSN 2375-2548.