Sweswi†Adirese ya sweswi: OX11 0DE, UK, Muako wa Dayimani, Phaka ya Sayense na Vumaki ya Harwell, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Senthara ya Swifaniso swa Swilo leswi hanyaka swa Elektroniki.
Reaction center light-harvesting complex 1 (RC-LH1) i xiphemu xa nkoka xa photosynthetic xa ti bacteria ta purple phototrophic. Hi nghenise swivumbeko swimbirhi swa cryo-electron microscopy swa RC-LH1 complex kusuka eka Rhodopseudomonas palustris. Xivumbeko xa 2.65-Å resolution xa RC-LH114-W complex xi na 14 wa ti subunit LH1 loops leti rhendzeleke RC, leti kavanyetiwaka hi protein W, kasi complex leyi nga riki na protein-W i xivumbeko xa RC hi ku helela lexi rhendzeriweke hi RC. Ku pfariwile 16 subunit LH1 loop. Ku pimanisiwa ka swivumbeko leswi swinyika vutivi eka dynamics ya quinone eka RC-LH1 complex, kukatsa na ku cinca ka conformational lokunga tivekiki khale loko ku bohiwa quinone eka ndzhawu ya RC QB, xikan’we na ndzhawu ya tindzhawu ta auxiliary quinone bonding sites, leswi pfunetaka Ku ti Hundzisela eka RC. Xivumbeko xo hlawuleka xa W protein xi sivela ku pfala ka LH1 loop, xisweswo xi endla channel yo hatlisisa ku cincana ka quinone/quinolone.
Matimba lama nyikiwaka hi photosynthesis ma nga hlayisa kwalomu ka vutomi hinkwabyo emisaveni, naswona ma ni vuswikoti lebyikulu eka thekinoloji ya swilo leswi hanyaka ya dyambu. Loko tiri karhi ti hlohletela photosynthesis ya misava hinkwayo, ti bacteria ta purple phototrophic titlhela ti kombisa tindlela to hambana hambana ta matimba na vuswikoti bya metabolic. Tinga papalata photosynthesis naswona tikula tani hi ti bacteria ta heterotrophic emunyameni, tinga lulamisa nitrogen na carbon dioxide, ti humesa hydrogen, naku onha ti aromatic compounds (1-3). Leswaku ku nyikiwa matimba eka maendlelo lawa, ku vonakala ku fanele ku hundzuriwa hi ku hatlisa ni hi ndlela leyinene ku va matimba ya tikhemikhali. Endlelo leri risungula loko light-trapping antenna complex yi tswonga ku vonakala naswona yi hundzisela eneji leyi khomiweke kuya eka reaction center (RC), xisweswo yisungula ku hambanisiwa ka charge (4 – 7). Yuniti ya xisekelo ya photosynthesis eka ti bacteria ta purple phototrophic yi vumbiwa hi muxaka wa 2 wa RC, lowu rhendzeriweke hi light-harvesting complex 1 (LH1), leswi endlaka RC-LH1 core complex. LH1 yi vumbiwa hi nxaxamelo wa ti heterodimers ta αβ leti govekeke, yin’wana na yin’wana ya tona yi boha ti molecule timbirhi ta bacterial chlorophyll (BChl) a na carotenoids yin’we kumbe timbirhi (8-12). Antena yo olova swinene ya LH1 yi vumbiwa hi 16 kumbe 17 wa ti-αβ heterodimers leti rhendzeleke RC (9-13) eka xirhendzevutana lexi pfalekeke, kambe eka ti-core complex tin’wana, ti- peptide ta transmembrane ti kavanyeta ku ya emahlweni ka LH1 leyi rhendzeleke , Hi ndlela yoleyo ti tlakusa ku hangalaka ka quinol/quinone exikarhi ka RC na cytochrome bc11 complex (11, 13-15). Ximilani xa purple phototrophic Rhodopseudomonas (Rps.) i xivumbiwa xa xikombiso lexi kotaka ku twisisa ku hundziseriwa ka matimba na ti electron leswi seketelaka photosynthesis. Xivumbeko xo sungula xa kristala xa Rps. Modele wa palustris RC-LH1 complex i RC, leyi rhendzeriweke hi 15 wa ti heterodimeric LH1 loops, leti kavanyetiwaka hi protein leyi nga tiviwiki leyi vuriwaka “Protein W” (14). Endzhaku ka sweswo protein-W yi tiviwile tani hi RPA4402, leyi nga protein leyi nga tivekiki ya 10.5kDa leyi nga na ti transmembrane helices tinharhu leti vhumbhiweke (TMH) ( 16 ). Hi ringanyeta ku thya vito lerintshwa ra rpa4402 gene leyi khodaka protein W ku va pufW leswaku yi fambisana na mavito lama tirhisiwaka eka tijini leti khodaka RC-L, M (pufL, pufM) na LH1α, β (pufA, pufB) subunits. Lexi tsakisaka, protein-W yikona ntsena eka kwalomu ka 10% wa RC-LH1, leswi paluxaka Rps. palustris yi humesa ti RC-LH1 complex timbirhi to hambana. Laha, hi vika swivumbeko swa high-resolution cryo-EM (cryo-EM) swa ti core complexes timbirhi, yin’wana yina protein W na 14 αβ heterodimers, yin’wana yina protein W na 16 Heterodimer LH1 loop leyi pfalekeke. Xivumbeko xa hina xi yimela ku cinca ka goza eka ku twisisa ka RC-LH1 complex ya Rps. palustris, hikuva hi kambisisile nhlayo ya homogeneous ya muxaka wun’wana na wun’wana naswona hi na xiboho lexi eneleke ku avela kahle peptide yin’wana na yin’wana na ti pigments leti bohiweke na ti lipids na ti quinones leti fambelanaka. Ku pimanisiwa ka swivumbeko leswi swikombisa leswaku ti protein tinharhu ta TMH-W letinga kumekaka eka RC-LH1 complex yin’wana na yin’wana kufikela sweswi ti humesa xiteji xa quinone ku hatlisisa ku cincana ka quinone/quinolone. Nhlayo ya tindzhawu leti hlayisiweke to boha lipid na quinone ti tiviwile, naswona hi paluxe ku cinca lokuntshwa ka conformational endzhaku ka ku hlanganisiwa ka quinone na RC, leswinga lulamelaka eka photosystem II (PSII) RC ya swivumbiwa swa phototrophic leswingana oxygen. Swikumiwa swa hina swi nyika vutivi byintshwa eka kinetics ya ku boha na ku cincana ka quinone/quinolone eka RC-LH1 core complex ya tibakteriya ta purple phototrophic.
Leswaku ku fambisiwa dyondzo ya vuxokoxoko bya ti complex timbirhi leti kumekaka eka Rps. palustris, hi hambanyisa RC-LH1 yin’wana na yin’wana hi tindlela ta biochemical. Xiphepherhele lexi pfumalaka tiphrotheyini W (laha ku nga ta vuriwa ΔpufW) xi basisiwile ku suka eka muxaka lowu pfumalaka jini ya pufW ( 16 ), naswona i xivumbeko xin’we ntsena xa RC-LH1 lexi nga humelerisiwaka. Complex leyingana protein W yi endliwa hi muxaka. Protein W ya muxaka lowu yi cinciwile hi 10x His tag eka C-terminus ya yona, leswaku complex leyingana protein W yinga hlanganisiwa kahle na protein W yotala leyi pfumalaka hiku yimisa nsimbi. Xivumbeko lexi xi hambanisiwile hi ndlela leyinene (16) Affinity Chromatography (IMAC).
Hilaha swi kombisiweke hakona eka Xifaniso xa 1, ti complexes leti hatimbirhi ti na ti sub-unit tinharhu ta RC (RC-L, RC-M na RC-H) leti rhendzeriweke hi antenna ya LH1. Xivumbeko xa 2.80-A xa complex leyi pfumalaka protein-W xi kombisa 16 wa ti αβ heterodimers, leti vumbaka LH1 loop leyi pfalekeke leyi rhendzeleke RC hi ku helela, laha endzhaku yi vuriwaka RC-LH116 complex. Xivumbeko xa 2.65Å xa protein-W-containing complex xina 14-heterodimer LH1 leyi kavanyetiweke hi protein-W, laha endzhaku yi vuriwaka RC-LH114-W.
(A na B) Vuyimeri bya le henhla bya nkatsakanyo. (C na D) Mihlovo leyi hlanganisiweke leyi kombisiweke hi tinhonga. (E na F) Ti complexes leti voniweke kusuka eka cytoplasmic surface tina ti peptides na ti LH1 subunits leti yimeriweke eka ti cartoons, naswona ti nomboriwile hi clockwise kusuka eka protein-W gap [leswi fambelanaka na Rba numbering. swiphepherhele swa sphaeroides (13)]. Eka LH1-α, muhlovo wa subunit ya ti protein i wo tshwuka; eka LH1-β, muvala wa subunit ya ti protein i wa wasi; eka protein-W, protein yi tshwuka; eka RC-H, i cyan; eka RC-L, i ya Orange; eka RC-M, ya magenta. Ti cofactors ti yimeriwe hi ti rods, rihlaza riyimela ti molecules ta BChl na BPh a, purple riyimela ti carotenoids, naswona yellow yiyimela ti molecules ta UQ10. (G na H) Xivono lexi kurisiweke xa xivandla xa tiphrotheyini-W eka ndhawu leyi ringanaka ya RC-LH114-W complex (G) na RC-LH116 complex (H). Ti cofactors ti kombisiwa hi xivumbeko xa space filling, chelated quinone yi kombisiwa hi muhlovo wa wasi. Xivandla xa protein-W xi kombisiwa hi layini ya wasi leyi nga na swiphemuphemu eka (G), naswona tiphuphu letintsongo laha quinone/quinolol yi hangalakaka eka ring ya LH116 ti kombisiwa hi layini ya ntima leyi nga na swiphemuphemu eka (H).
Xifaniso xa 1 (A na B) xi kombisa RC leyi rhendzeriweke hi ti arrays leti pfulekeke kumbe leti pfalekeke ta LH1αβ heterodimers, yin’wana na yin’wana ya tona yi boha BChl timbirhi na carotenoid yin’we (Xifaniso 1, C na D). Mindzavisiso ya khale yi kombisile leswaku Rps i LH1 complex. Eka ndlela ya biosynthetic ya spirulina xanthin, tinxaka leti tina nhlayo leyi pfanganisiweke ya ti carotenoids ( 17 ). Hambiswiritano, spiropyrroxanthin hi yona carotenoid leyikulu naswona density ya yona ya eneriseka. Hikwalaho, hi hlawurile ku modela spiroxanthin eka tindhawu hinkwato to boha LH1. Ti alpha na beta polypeptides i ti TMH tin’we letingana tindzhawu to koma ta membrane yale handle (Xifaniso 1, A, B, E, na F). Hambi leswi density ya 17 wa masalela eka C-terminus yinga voniwa, alpha polypeptide yi hambanisiwile kusuka eka Met1 kuya eka Ala46 eka ti complexes leti hatimbirhi. β polypeptide yi hungutiwile kusuka eka Gly4 kuya eka Tyr52 eka RC-LH116, na kusuka eka Ser5 kuya eka Tyr52 eka RC-LH114-W. Ku hava density ya 3 kumbe 4 wa N-terminal kumbe 13 wa masalela ya C-terminal leyi voniweke (Xifaniso xa S1). Nxopaxopo wa mass spectrometry wa mixed RC-LH1 complex leyi lulamisiweke kusuka eka muxaka wa nhova wu kombisile leswaku ndzhawu leyi kayivelaka akuri mbuyelo wa heterologous cleavage ya ti peptides leti (Figure S1 na S2). N-terminal formylation ya α-Met1 na yona yi voniwile (f). Nxopaxopo wu kombisile leswaku α-peptide yi vumbiwa hi masalela ya fMet1 ku ya eka Asp42/Ala46/Ala47/Ala50, naswona β-peptide yi vumbiwa hi masalela ya Ser2 ku ya eka Ala53, leswi pfumelelanaka kahle na mepe wa EM density ya mahiselo ya le hansi.
Ku hlanganisiwa ka α-His29 na β-His36 swi endla leswaku BChls yi langutana na xikandza; αβ heterodimer yin’wana na yin’wana yi hlangana na vaakelani va yona ku vumba xirhendzevutana lexi pfulekeke (RC-LH114-W) kumbe xirhendzevutana lexi pfalekeke (RC-LH116) ku rhendzela RC The exciton coupled pigment array (Xifaniso 1, C na D). Loko ku pimanisiwa na 877 nm band ya RC-LH114-W, 880 nm absorption red shift ya RC-LH116 i 3 nm (Xifaniso 2A). Hambiswiritano, spectrum ya circular dichroism yi lava ku fana (Figure 2B), leswi kombisaka leswaku hambi leswi kungana ku hambana loku nga erivaleni exikarhi ka ti loop leti pfulekeke na leti pfalekeke, ndzhawu ya ndzhawu ya BChls yafana swinene. Ku cinca ka muhlovo wo tshwuka ka ku tswongiwa ku nga ha va ku ri vuyelo bya ku hunguteka ka ku famba ka mahiselo ni ku engeteleka ka ku tiya eka xirhendzevutana lexi pfalekeke (18, 19), ku cinca ka ku hlanganisiwa ka muhlovo loku vangiwaka hi xirhendzevutana lexi pfalekeke (20, 21), kumbe ku hlanganisiwa ka switandzhaku leswi swimbirhi (11).
(A) Ultraviolet/visible/near-infrared absorption spectrum, tinhlohlorhi ta yona ti funghiwile hi ti pigments ta tona leti fambelanaka naswona ti normalized eka nhlohlorhi ya BPh eka 775 nm. (B) Xirhendzevutani xa dichroism spectrum normalized eka BChl absorbance eka 805 nm. (C na D) Swiphemu swa ΔA leswi hlawuriweke ku suka eka swiphemu swa ku amukela leswi lulamisiweke hi nkarhi swa RC-LH114-W complex (C) na RC-LH116 complex (D). Kuva ku pimanisiwa ku antswa, ti spectra hinkwato ti normalised kuya eka ∆A ya −A eka 0.2 ps. (E) Mpimo wa cytochrome c2 oxidation endzhaku ka irradiation eka vukona bya ti concentrations to hambana ta UQ2 (vona Xifaniso xa S8 ku kuma data yo ka yi nga swekiwanga). (F) Eka tisele leti byariweke ehansi ka ku vonakala ka le hansi, ka le xikarhi kumbe ka matimba ya le henhla (10, 30 kumbe 300μMm-2 s-1, hi ku landzelelana), ti protein W na RC-L subunits eka complex leyi basisiweke na ratio ya membrane leyi hambanisiweke. Ku kumisisa mpimo wa tiphrotheyini hi SDS-polyacrylamide gel electrophoresis na immunoassay (vona Xifaniso xa S9 ku kuma datha yo ka yi nga swekiwanga). Kuma ratio loko yi pimanisiwa na RC-LH114-W complex leyi basisiweke. Nhlayo ya stoichiometric ya RC-L kuya eka protein-W ya complex i 1:1.
Ti BChls eka xiyimo xa 1 eka deformed αβ14 loop ya RC-LH114-W (Xifaniso 1, A, C, na E) ti le kusuhi na RC primary donor (P) hi 6.8Å ku tlula BChls leyi ringanaka eka RC-LH116 (Xifaniso. 1, B, D, na F, na Xifaniso S3); hambiswiritano, ti kinetics ta transient absorption ta ti complex timbirhi ti kombisa leswaku eka RC-LH114-W na RC-LH116, ti constants ta nkarhi wo hundzisela eneji ya excitation kusuka eka LH1 kuya eka RC i 40 ±4 na 44±3 ps (Xifaniso 2). , C na D, Xifaniso xa S4 na Tafula ra S2). Nakambe a ku na ku hambana lokukulu eka ku hundziseriwa ka xielekitironiki endzeni ka RC (Xifaniso xa S5 na tsalwa ro engetela leri fambelanaka). Hi sola leswaku ku fambelana lokukulu ka nkarhi wa ku hundziseriwa ka eneji exikarhi ka LH1 na RC-P swi vangiwa hi mpfhuka lowu fanaka, angle na eneji leyi nga kona ya BChl yo tala eka swirhendzevutana swimbirhi swa LH1. Switikomba onge ku lavisisa xivumbeko xa eneji ya LH1 ku fikelela mpfhuka wale hansi aswi hatlisi kutlula ku hundziseriwa ka eneji hiku kongoma kusuka eka tindzhawu letinga suboptimal kuya eka RC. Open-loop LH1 loop eka RC-LH114-W yinga tlhela yi hundza eka ku famba ka mahiselo lokunga nyawuriki ehansi ka swiyimo swa mahiselo yale hansi eka nxopaxopo wa xivumbeko, naswona kuna αβ14 ring conformation yo leha eka mahiselo ya kamara kusuka eka mpfhuka wa pigmentation wa βBChls eka xiyimo xa RC 1.
RC-LH116 complex yina 32 wa ti BChls na 16 wa ti carotenoids, naswona maendlelo ya yona hinkwawo yafana na lawa ya kumiweke kusuka eka Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 strain ( PDB ID 7C9R) (12) na rihlaza switsotswana leswi vuriwaka algae (Blc.viridis) (ID ya PDB 6ET5) (10). Endzhaku ka ku ringanisiwa, ku voniwe ntsena ku hambuka lokuntsongo eka swiyimo swa αβ heterodimers, ngopfu ngopfu 1-5, 15, na 16 (Xifaniso xa S6). Kuva kona ka protein-W swina nkucetelo lowukulu eka xivumbeko xa LH1. Ti TMH ta yona tinharhu ti hlanganisiwa hi ti loops to koma, laha N-terminal yinga eka tlhelo ra lumen ra complex na C-terminal eka tlhelo ra cytoplasmic (Swifaniso 1A na 3, A kuya eka D). Protein-W ngopfu ngopfu yi na hydrophobic (Xifaniso 3B), naswona TMH2 na TMH3 swi tirhisana na LH1αβ-14 ku endla vuandlalo bya transmembrane (Xifaniso 3, B na E ku ya eka G). Interface yivumbiwa ngopfu hi masalela ya Phe, Leu na Val eka ndzhawu ya transmembrane. Masalela lawa ya hlengeletiwile na ti hydrophobic amino acids na ti αβ-14 pigments. Masalela yan’wana ya polar yatlhela ya hoxa xandla eka ku tirhisana, kukatsa na hydrogen bond exikarhi ka W-Thr68 na β-Trp42 ehenhla ka complex cavity (Figure 3, F na G). Ehenhla ka cytoplasm, Gln34 yi le kusuhi na ntlawa wa keto wa αβ-14 carotenoids. Ku engetela kwalano, molecule ya n-dodecyl β-d-maltoside (β-DDM) yi lulamisiwile, naswona ncila wa yona wa hydrophobic wu andlariwile kuya eka interface exikarhi ka protein-W na αβ-14, naswona ncila wa lipid wunga kumeka emirini. Hi tlhele hi xiya leswaku tindzhawu ta C-terminal resolution ta protein W na RCH ti le kusuhi swinene, kambe kungari endzeni ka xikalo xo vumba ku tirhisana ko karhi (Xifaniso 1, A na E). Hambiswiritano, kungava na ku tirhisana eka ti amino acids ta C-terminal letinga lulamisiwangiki ta ti protein leti timbirhi, leswinga nyikaka endlelo ro thoriwa ka protein-W hi nkarhi wa nhlengeletano ya RC-LH114-W complex.
(A) Protein-W, leyi langutaneke na ku hlangana na LH1αβ14 hi xivumbeko xa khathuni, yi na xivumbeko xa nhonga xa nketani ya le tlhelo (yo tshwuka), leyi kombisiweke eka xiphemu xa xifaniso xa vuswikoti bya electrostatic (vuandlalo bya grey lebyi nga erivaleni lebyi nga na mpimo wa contour wa 0.13). (B) Protein-W leyi yimeriwaka hi xivumbeko xa muvala wa hydrophobic. Tindzhawu ta polar na charged ti kombisiwa hi cyan, tindzhawu letinga hydrophobic ti kombisiwa hi muhlovo wo basa, naswona tindzhawu letinga na hydrophobic swinene ti kombisiwa hi muhlovo wa orenji. (C na D) Protein-W leyi yimeriweke eka khathuni, ku kongoma ka yona ku fana na le ka (A) (C), naswona yi rhendzeleka hi 180° (D). Kuya hi xiyimo eka ntlhandlamano, masalela lawa ya hambanisiwaka ya amukela xikimi xa mihlovo ya ntima, laha N-terminal yinga ya wasi kasi C-terminal yinga yotshuka. (E) Protein-W hi ku vona loku fanaka na le ka (A), naswona masalela eka interface ya protein-W:LH1 ya yimeriwile hi tinhonga leti nga na swikoweto leswi khomanisiweke. (F) Protein-W yi rhendzeleka hi 90° loko yi pimanisiwa na (E) na LH1αβ14 eka vuyimeri bya khathuni, naswona yi pimanisiwa na masalela ya interface eka vuyimeri bya bar. Masalela lawa ya le henhla kusuka eka beta polypeptide ya tsariwile. Cofactor yi kombisiwile tani hi bar leyi fambelanaka na muhlovo wa Figure 1, β-DDM leyi boleke yi kombisiwile hi grey, naswona oxygen yi kombisiwile hi muhlovo wo tshwuka. (G) Xivono lexi nga eka (F) xi rhendzelekile hi 180°, na masalela lama vonakaka ya alpha polypeptide leyi leteriweke.
Protein-W yi siva αβ heterodimer (ya vu 15 eka Xifaniso xa 1F), hi ndlela yaleyo yi sivela ku pfala ka loop naku tilta ti αβ heterodimers tinharhu tosungula. Ku xiyiwile leswaku angle ya le henhla ya inclination ya heterodimer yo sungula ya αβ-1 loko yi pimanisiwa na film normal a yi ri 25° ku ya eka 29° (Xifaniso 1, A na E), leswi vumbiweke hi 2° ku ya eka 8° inclination ya αβ-1 eka RC A sharp contrast-LH116 (Xifaniso 1, B na F). Heterodimers ya vumbirhi na ya vunharhu yi rhendzelekile eka 12° kuya eka 22° na 5° kuya eka 10°, hiku landzelelana. Hikwalaho ka xihinga xa steric xa RC, ku tilt ka αβ-1 a ku katsi mpatswa wa vumbirhi wa αβ (lowu fambelanaka na αβ ya vu 16 eka Xifaniso 1F), xisweswo ku vumba xivandla lexi nga erivaleni eka ring ya LH1 (Xifaniso 1, A na E) . Hikwalaho ka ku pfumaleka ka ti αβ heterodimers timbirhi, leswi fambisanaka na ku lahlekeriwa hi mune wa BChl na ti carotenoids timbirhi, ku hava na yin’we ya ti carotenoids leyi bohaka eka twisted αβ-1 subunit, leswi endlaka leswaku ku va na ring ya LH114-W leyi nga na 13 wa ti carotenoids Vegetarian na 28 wa ti BChls. Swiringanyeto swa xiboho xa laha kaya swa swivumbeko swimbirhi eka swifundzha swa αβ1 ku ya eka 7 swi le hansi ku tlula swa LH1 loop hinkwayo, leswi nga kombisaka pulasitiki ya ntumbuluko ya subunit ya LH1 leyi nga ekusuhi na ndhawu ya RC QB (Xifaniso 4) .
Swifaniso swa RC-LH114-W (A na B) na RC-LH116 (C na D) swi kombisiwile ku suka eka xivono xa le henhla/xivono xa le tlhelo (A na B) (A na C) na vuandlalo bya cavity bya Fig. 1. (B na D). Swilotlelo swa mihlovo swi kombisiwile etlhelo ra xinene.
Xihlawulekisi xin’wana ntsena xa core complex lexi nga na stoichiometric ratio ya 1:14 i Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Hambiswiritano, protein W na PufX a swi na homology leyi nga erivaleni, naswona swi na nkucetelo lowukulu eka swivumbeko swa swona swa LH1 hi ku hambana ka swona. PufX i TMH yin’we leyi nga na N-terminal cytoplasmic domain leyi tirhisanaka na tlhelo ra cytoplasmic ra RC-H subunit (13) eka xiyimo lexi fambelanaka na Rps. palustris LH116αβ-16. PufX yi tumbuluxa xiteji xa ku cincana ka quinone/quinolone exikarhi ka RC-LH1 na cytochrome bcl complex naswona yi kona eka Rba hinkwayo. sphaeroides xirhendzevutani xa le xikarhi (13). Hambi leswi xihlanganisi xa monomer-monomer xi nga eka Rba. Sphaeroides RC-LH1-PufX dimer yi kumeka eka xiyimo xo boha xa protein W eka RC-LH114-W, naswona xivandla lexi hlohloteriwaka hi PufX na protein-W xi kumeka eka xiyimo xo ringana (Xifaniso S7A). Xivandla eka RC-LH114-W xitlhela xi fambelanisiwa na xiteji xa quinone xa xiringanyeto (8) xa Pseudomonas rosea LH1, lexi vumbiwaka hi ti peptides letinga fambelaniki na protein W kumbe PufX (Xifaniso S7B). Ku engetela kwalaho, xiteji xa quinone eBlc. LH1 ya rihlaza ra emerald leyi vumbiweke hi ku susa subunit yin’we ya γ (7) yi kumeka eka xiyimo lexi fanaka (Xifaniso xa S7C). Hambi leswi swi kongomisiwaka hi ti protein to hambana, ku humelela ka ti quinone/quinolol channels leti eka xiyimo lexi fanaka eka RC-LH1 complex switikomba swiri xikombiso xa convergent evolution, leswi kombaka leswaku xivandla lexi tumbuluxiweke hi protein W xinga tirha tani hi quinone channel.
Xivandla eka LH114-W loop xi pfumelela ku tumbuluxiwa ka ndzhawu ya membrane leyi yaka emahlweni exikarhi ka ndzhawu yale ndzeni ya RC-LH114-W complex na bulk membrane (Figure 1G), kutlula ku hlanganisa ti domains timbirhi hiku tirhisa protein pore tani hi le ka ti proteins. Xivumbeko xa RC-LH116 xi fana na Tch leyi pfalekeke. Xivumbeko lexi fanaka ni naliti (22) (Xifaniso 1H). Leswi ku hangalaka ka quinone hiku tirhisa membrane swi hatlisaka kutlula ku hangalaka hiku tirhisa channel channel yo koma, LH114-W loop leyi pfulekeke yinga pfumelela ku cinca ka RC hiku hatlisa kutlula LH116 loop leyi pfalekeke, naswona ku hangalaka ka quinone eka RC swinga siveriwa swinene. Ku kota ku kambela loko protein W yi khumba ku hundzuriwa ka ti quinones hiku tirhisa RC, hi endlile cytochrome oxidation assay eka nhlayo yokarhi ya ubiquinone 2 (UQ2) (analog ya ntumbuluko ya UQ10 leyingana ncila wo koma wa isoprene) (Xifaniso 2E). Hambi leswi vukona bya chelated quinone byi sivelaka ku kumiwa lokunene ka Michaelis constant leyi vonakaka (RC-LH114-W na RC-LH116 swi lulamerile eka 0.2±0.1μM na 0.5±0.2μM, hi ku landzelelana), mpimo wa le henhla wa RC-LH114-W ( 4.6±0.2 e-RC-1 s-1) i 28±5% yikulu kutlula RC-LH116 (3.6±0.1 e-RC-1 s-1).
Eku sunguleni hi ringanyete leswaku protein-W yi kona eka kwalomu ka 10% wa core complex (16); laha, nhlayo ya ku tshama ka ti cells ta ku kula ka ku vonakala ka le hansi, ku vonakala ka le xikarhi, na ku vonakala lokukulu i 15±0.6%, 11±1% na 0.9±0.5, hi ku landzelelana % (Xifaniso 2F). Ku pimanisiwa ka nhlayo ya mass spectrometry swikombe leswaku ku engeteriwa ka histidine tag aswi hungutanga nhlayo ya relative ya protein-W loko ku pimanyisiwa na tinxaka ta muxaka wa nhova (P = 0.59), hikokwalaho ti levele leti ahi artifact ya modified protein-W (Figure S10). Hambiswiritano, ku tshama loku ka le hansi ka protein-W eka RC-LH1 complex ku nga pfumelela ti RC tin’wana ku flip hi rivilo leri hatlisisiweke, hi ndlela yaleyo ku hunguta ku cincana ka quinone/quinolone loku nonokaka eka RC-LH116 complex. Hi xiye leswaku mpimo wa le henhla wa ku tshama ka ku vonakala a wu fambisani na datha ya sweswinyana ya transcriptomics, leswi kombisaka leswaku ku paluxiwa ka tijini ta pufW ku tlakuka ehansi ka ku vonakala lokukulu (Xifaniso S11) (23). Ku hambana exikarhi ka pufW transcription na ku nghenisiwa ka protein-W eka RC-LH1 complex swa pfilunganya naswona swinga kombisa vulawuri byo rharhangana bya protein.
Eka RC-LH114-W, 6 wa cardiolipin (CDL), 7 wa phosphatidylcholine (POPC), 1 wa phosphatidylglycerol (POPG) na 29 wa timolekhuli ta β-DDM ti averiwe no modeliwa eka yona 6 wa ti-CDL, 24 wa ti-POPC, 2 wa ti-POPG na 12 wa ti-βDDM. RC-LH116 (Xifaniso xa 5, A na B). Eka swivumbeko leswi swimbirhi, CDL yi lava ku kumeka eka tlhelo ra cytoplasmic ra complex, kasi POPC, POPG na β-DDM swi kumeka ngopfu eka tlhelo ra luminal. Timolekhuli timbirhi ta lipid na detergent ti hambanisiwile eka ndzhawu ya αβ-1 kuya eka αβ-6 ya RC-LH114-W complex (Xifaniso 5A), naswona ntlhanu ti hambanisiwile eka ndzhawu leyi ringanaka ya RC-LH116 (Xifaniso 5B ). Ti lipids totala tikumeke eka tlhelo rin’wana ra complex, ngopfu ngopfu CDL, leyi hlengeletiweke exikarhi ka RC na αβ-7 kuya eka αβ-13 (Xifaniso 5, A na B). Ti lipids tin’wana leti lulamisiweke hi xivumbeko na ti detergent ti kumeka ehandle ka ring ya LH1, naswona ti acyl chains leti lulamisiweke kahle ti anama exikarhi ka ti subunits ta LH1, leti hlamuseriweke hi xihatla tani hi β-DDM eka RC-LH114-W, naswona ti hlamuseriwa tani hi β-DDM eka RC Nkatsakanyo wa β-DDM na POPC-LH116. Swiyimo leswi fanaka swa ti chelating lipids na ti detergents eka xivumbeko xa hina swikomba leswaku i tindzhawu to boha leti fambelanaka na physiologically (Xifaniso xa S12A). Swiyimo swa ti molecule leti ringanaka eka Tch na swona swina ku ringanana lokunene. Ku olova na Trv. Muxaka wa 970 RC-LH1s (Xifaniso S12, B kuya eka E) (9, 12) na masalela ya hydrogen-bonding ya ntlawa wa nhloko ya lipid swikombe nhlayiso lowunene swinene eka ku ringanisiwa ka ntlhandlamano (Xifaniso S13), leswi kombisaka leswaku Conserved CDL leyi bohaka eka RC (24), tindzhawu leti tinga hlayisiwa eka RC-LH1 complex.
(A na B) Tipeptide ta RC-LH114-W (A) na RC-LH116 (B) ti yimeriwe hi tikhathuni, naswona ti pigment ti yimeriwe hi tinhonga, hi ku tirhisa xikimi xa mihlovo eka Xifaniso 1. Tilipid ti kombisiwile hi muhlovo wo tshwuka, naswona swibasisi swi kombisiwile hi grey. UQ leyi bohiweke eka tindzhawu ta RC QA na QB yina muhlovo wotshuka, kasi UQ leyi nga yoxe i ya wasi. (C na D) Mavonelo lama fanaka na (A) na (B), laha ku tshikiwile ti lipids. (E ku ya eka G) Xivono lexi kurisiweke xa Q1(E), Q2(F) na Q3(G) ku suka eka RC-LH116, na tikhetane ta le matlhelo leti khumbhanaka. Swihlanganisi swa hayidirojeni swi kombisiwa tanihi milayeni ya ntima leyi nga ni swiphemuphemu.
Eka RC-LH116, havumbirhi bya RC QA na QB UQ, leswi nghenelaka eka ku hundziseriwa ka tielektroni eka endlelo ro hambanyisa charge, swi bola eka tindzhawu ta swona to boha. Hambiswiritano, eka RC-LH114-W, QB quinone a yi lulamisiwanga naswona yi ta xopaxopiwa hi vuxokoxoko laha hansi. Ku engetela eka QA na QB quinones, ti molecule timbirhi ta UQ leti chelated (leti kumekaka exikarhi ka ti ring ta RC na LH1) ti averiwe eka xivumbeko xa RC-LH114-W kuya hi mintlawa ya tona ya tinhloko leyi lulamisiweke kahle (leyi kumekaka eka Q1 na Q2, hiku landzelelana). xivandla). Xifaniso xa 5C). Tiyuniti timbirhi ta isoprene ti averiwe Q1, naswona mepe wa density wu lulamisa misisi ya 10 leyi heleleke ya isoprene ya Q2. Eka xivumbeko xa RC-LH116, ti molecule tinharhu ta UQ10 leti chelated (Q1 kuya eka Q3, Figure 5D) ti lulamisiwile, naswona ti molecule hinkwato tina density leyi nga erivaleni eka ncila hinkwawo (Figure 5, D kuya eka G). Eka swivumbeko leswimbirhi, swiyimo swa mintlawa ya tinhloko ta quinone ta Q1 na Q2 swina ku ringanana lokunene (Xifaniso S12F), naswona swi tirhisana ntsena na RC. Q1 yi kumeka eka nyangwa wa W gap ya RC-LH114-W (Xifaniso 1G na 5, C, D na E), naswona Q2 yi kumeka ekusuhi na ndzhawu yo boha ya QB (Xifaniso 5, C, D) na F). Masalela ya L-Trp143 na L-Trp269 lawa ya hlayisiweke ya le kusuhi swinene na Q1 na Q2 naswona ya nyika ku tirhisana loku nga vaka kona ka π-stacking (Xifaniso 5, E na F, na Xifaniso xa S12). L-Gln88, 3.0 Å kusuka eka okisijini yale kule ya Q1, yinyika vuxaka byo tiya bya hayidirojeni (Xifaniso 5E); masalela lawa ya hlayisiwa eka ti RC hinkwato handle ka vuxaka bya le kule swinene (Xifaniso xa S13). L-Ser91 yi siviwe hi ndlela yo hlayisa Thr eka ti RC tin’wana totala (Xifaniso S13), i 3.8 Angstroms kusuka eka methyl oxygen ya Q1, naswona yinga nyika swihlanganisi swa hydrogen leswinga tsana (Xifaniso 5E). Q3 ayi vonaki yiri na ku tirhisana kokarhi, kambe yi kumeka eka ndzhawu ya hydrophobic exikarhi ka RC-M subunit na LH1-α subunit 5 kuya eka 6 (Xifaniso 5, D na G). Q1, Q2 na Q3 kumbe ti chelated quinones letinga ekusuhi na tona ti lulamisiwile eka Tch. Ku olova, Trv. Xiphemu xa 970 na Blc. Xivumbeko xa iris (9, 10, 12) xi kombisa ndzhawu yo boha ya quinone yo pfuneta leyi hlayisiweke eka RC-LH1 complex (Xifaniso S12G). Ti UQ ta ntlhanu leti boleke eka RC-LH116 ti pfumelelana kahle na 5.8±0.7 ya complex yin’wana na yin’wana leyi kumiweke hi high performance liquid chromatography (HPLC), kasi ti UQ tinharhu leti boleke eka RC-LH114-W ti le hansi ka Nhlayo leyi pimiweke ya 6.2±0.3 (Fig. S14) yi kombisa leswaku ku na ti molecule ta UQ leti nga tlhantlhiwangiki eka xivumbeko.
Ti polypeptides ta pseudo-symmetric L na M yin’wana na yin’wana yina ntlhanu wa ti TMHs naswona yi endla heterodimer leyi hlanganisaka BChl dimer yin’we, ti BChl monomers timbirhi, ti bacteriophage (BPh) monomers timbirhi, na yin’we leyi nga riki ya Heme iron na molecule yin’we kumbe timbirhi ta UQ10. Hikuva na vukona bya hydrogen bonds eka ntlawa wa terminal ketone xikan’we naku hlengeletiwa ka yona loku tivekaka eka Rps, ti carotenoids ti nghenisiwa eka M-subunit, leyi thyiweke cis-3,4-dehydroorhodopin. Tinxaka ta swiharhi (25). Domain ya membrane yale handle ya RC-H yi khomiwile eka membrane hi TMH yin’we. Xivumbeko xa RC hinkwaxo xifana na ti subunit tinharhu ta RC ta tinxaka leti fambelanaka (kufana na Rba). ti- sphaeroides (ID ya PDB: 3I4D). Ti macrocycles ta BChl na BPh, carotenoid backbone na non-heme iron swi hlangana endzeni ka resolution range ya swivumbeko leswi, tani hileswi swiendlaka eka ntlawa wa nhloko ya UQ10 eka ndzhawu ya QA na QB quinone eka RC-LH116 (Xifaniso xa S15).
Ku kumeka ka swivumbeko swimbirhi swa RC leswi nga na mimpimo yo hambana ya ku tshama ka ndhawu ya QB swi nyika nkarhi wuntshwa wo kambisisa ku cinca ka conformational loku nga cincekiki loku fambisanaka na ku bohiwa ka QB quinone. Eka RC-LH116 complex, QB quinone yi kumeka eka xiyimo xa “proximal” lexi bohiweke hi ku helela ( 26 ), kambe ku hambanisiwa ka RC-LH114-W a ku na QB quinone. Akuna QB quinone eka RC-LH114-W, leswi hlamarisaka hikuva complex ya tirha, kutlula RC-LH116 complex leyingana structurally resolved QB quinone. Hambi leswi ti ring timbirhi ta LH1 ti chelate kwalomu ka tsevu wa ti quinones, ntlhanu ti lulamisiwile hi xivumbeko eka ring ya RC-LH116 leyi pfalekeke, kasi tinharhu ntsena ti pimiwile hi xivumbeko eka ring leyi pfulekeke ya RC-LH114-W . Ku tlakuka loku ka ku pfilunganyeka ka xivumbeko ku nga ha kombisa ku hatlisa ku siviwa ka tindhawu ta RC-LH114-W QB, ku hatlisa ka quinone kinetics eka complex, na ku engeteleka ka ku koteka ka ku tsemakanya LH1 loop. Hi ringanyeta leswaku ku pfumaleka ka UQ eka ndzhawu ya RC QB ya RC-LH114-W swinga va mbuyelo wa complex leyi rharhanganeke swinene na leyi tirhaka swinene, naswona ndzhawu ya QB ya RC-LH114-W yi hatle yi titimela eka ku cinca ka UQ Xiteji xokarhi (ku nghena eka ndzhawu ya QB ku pfariwile) xi kombisa conformation ya ntirho lowu.
Handle ka QB, ku rhendzeleka loku fambisanaka na L-Phe217 eka xiyimo lexi nga fambelaniki na ku bohiwa ka UQ10, hikuva swi ta vanga ku tlumbana ka ndhawu na yuniti yo sungula ya isoprene ya ncila (Xifaniso 6A). Ku engetela kwalaho, ku cinca lokukulu loku nga erivaleni ka conformational ku le rivaleni, ngopfu ngopfu helix de (helix yo koma eka loop exikarhi ka TMH D na E) laha L-Phe217 yi rhurhiseriwaka eka xikhwama xo boha xa QB na ku rhendzeleka ka L-Tyr223 (Xifaniso 6A ) Ku tshovela xihlanganisi xa hayidirojeni na xivumbeko xa M-Asp45 na ku pfala nyangwa wa ndhawu yo boha ya QB (Xifaniso xa 6B). Helix de pivots eka xisekelo xa yona, Cα ya L-Ser209 yi cinciwa hi 0.33Å, kasi L-Val221Cα yi cinciwa hi 3.52Å. Ku hava ku cinca loku vonakaka eka TMH D na E, loku nga superimposable eka swivumbeko leswimbirhi (Xifaniso 6A). Hi laha hi switivaka ha kona, lexi i xivumbeko xosungula eka RC ya ntumbuluko lexi pfala ndzhawu ya QB. Ku pimanisiwa na xivumbeko lexi heleleke (QB-bound) swikombisa leswaku loko quinone yinga se hungutiwa, ku cinca ka conformational swa laveka kuva yiva yi nghena eka quinone. L-Phe217 yi rhendzeleka ku vumba ku tirhisana ka π-stacking na ntlawa wa nhloko ya quinone, naswona helix yi cincela ehandle, leswi pfumelelaka skeleton ya L-Gly222 na side chain ya L-Tyr223 ku vumba network ya hydrogen bond leyi nga na xivumbeko xa hydrogen bond lexi tiyeke (Xifaniso 6, A na C).
(A) Khathuni leyi hlanganisaka ya hologram (L chain, orange/M chain, magenta) na xivumbeko xa apo (gray), laha masalela ya nkoka ya kombisiwaka hi xivumbeko xa xifaniso xo fana na nhonga. UQ10 yi yimeriwe hi bar yo tshwuka. Layini leyi nga ni mathonsi yi kombisa swihlanganisi swa hayidirojeni leswi vumbiweke eka xivumbeko hinkwaxo. (B na C) Vuyimeri bya le henhla bya apolipoprotein na xivumbeko hinkwaxo xa ring, ku kombisa okisijini ya ketane ya le tlhelo ya L-Phe217 hi muhlovo wa wasi na L-Tyr223 hi muhlovo wo tshwuka, hi ku landzelelana. Subunit ya L i ya muhlovo wa orenji; ti subunits ta M na H a ti na mihlovo. (D na E) Tindhawu ta apolipoprotein (D) na leti heleleke (E) ta RC QB [muhlovo hi (A) hi ku landzelelana] na Thermophilus thermophilus PSII (ya rihlaza, ya wasi na quinone ya pulasitiki; PDB ID: 3WU2) Lulamisa (58).
Swi nga languteriwanga, hambi leswi swivumbeko swo hlaya swa ti RC leti pfumalaka QB leti nga riki na LH1 swi kumekaka, ku cinca ka conformational loku voniweke eka ndzavisiso lowu a ku si vikiwa khale. Leswi swi katsa xivumbeko xa QB depletion kusuka eka Blc. viridis (ID ya PDB: 3PRC) (27), Tch. tepidum (ID ya PDB: 1EYS) (28) na Rba. sphaeroides (PDB ID: 1OGV) (29), hinkwaswo leswi swi lava ku fana na xivumbeko xa swona xa QB hinkwaxo. Ku kamberiwa hi vukheta ka 3PRC swi paluxe leswaku ti molecule ta detergent ta LDAO (Lauryl Dimethyl Amine Oxide) ti boha enyangweni wa xiyimo xa QB, leswi nga sivela ku lulamisiwa nakambe eka conformation leyi pfalekeke. Hambi leswi LDAO yinga bola eka ndzhawu yin’we eka 1EYS kumbe 1OGV, ti RCs leti ti lulamisiwa hiku tirhisa detergent yin’we naswona hikokwalaho tinga humesa mbuyelo lowu fanaka. Xivumbeko xa kristala xa Rba. Sphaeroides RC co-crystallized na cytochrome c2 (PDB ID: 1L9B) na yona yi vonaka yi ri na ndzhawu ya QB leyi pfalekeke. Kambe, eka xiyimo lexi, ndhawu ya N-terminal ya RC-M polypeptide (ku tirhisana na ndhawu yo boha ya QB hi ku tirhisa H bond ya masalela ya Tyr eka Q helix) yi amukela conformation leyi nga riki ya ntumbuluko, naswona ku cinca ka conformational ya QB a ku lavisisiwa ku ya emahlweni (30 ). Lexi tiyisisaka hileswaku a hi vonanga muxaka lowu wa ku onhaka ka M polypeptide eka xivumbeko xa RC-LH114-W, lexi nga kwalomu ka ku fana na ndhawu ya N-terminal ya RC-LH116 RC. Swi fanele ku tlhela swi xiyiwa leswaku endzhaku ka ku herisiwa ka antenna ya LH1 leyi simekiweke eka detergent, ti apolipoprotein RCs eka PDB ti lulamisiwile, leswi heriseke swidziva swa le ndzeni swa quinone na ti lipids eka xivandla exikarhi ka RC na vuandlalo bya le ndzeni bya ring ya LH1 leyi rhendzeleke ( 31 , 32 ). RC yi tshama yi ri karhi yi tirha hikuva yi hlayisa ti cofactors hinkwato, handle ka QB quinone leyi bolaka, leyi nga tshamisekangiki ngopfu naswona yi talaka ku lahleka hi nkarhi wa endlelo ro lunghiselela (33). Ku engetela kwalaho, swa tiveka leswaku ku susiwa ka LH1 na ti-lipid ta ntumbuluko ta xirhendzevutani eka RC swi nga va na nkucetelo eka mintirho, ku fana na ku hungutiwa ka vutomi bya xiyimo xa P+QB lexi hambanisiweke hi charge ( 31 , 34 , 35 ). Hikokwalaho, hi ringanyeta leswaku vukona bya ring ya ndzhawu ya LH1 leyi rhendzeleke RC swinga hlayisa ndzhawu ya QB leyi “pfaleriweke”, hindlela yaleyo ku hlayisa ndzhawu ya ndzhawu ekusuhi na QB.
Hambi leswi apolipoprotein (handle ka QB quinone) na xivumbeko lexi heleleke swiyimelaka ntsena swifaniso swimbirhi swa xihatla swa ku cinca ka ndzhawu ya QB, kutlula nxaxamelo wa swiendlakalo, kuna swikombiso swa leswaku ku bohiwa kunga gated ku sivela ku bohiwa nakambe hi hydroquinone Ku sivela substrate inhibition. Ku tirhisana ka quinolol na quinone ekusuhi na ndzhawu ya QB ya apolipoprotein swinga hambana, leswi yisaka eka ku ariwa ka yona hi RC. I khale swiringanyetiwa leswaku ku cinca ka conformational swina xiave eka ku boha naku hunguta ti quinones. Vuswikoti bya ti RC leti titimelaka ku hunguta ti quinones endzhaku ka ku tolovela munyama byi onhakile (36); X-ray crystallography yi kombisa leswaku ku onhaka loku ku vangiwa hi ti-QB quinones leti khomiweke eka “distal” conformation kwalomu ka 4.5 Å ku suka eka xiyimo xa le kusuhi lexi tirhaka (26) , 37). Hi ringanyeta leswaku conformation leyi ya distal binding i xifaniso xa xihatla xa xiyimo xale xikarhi exikarhi ka apolipoprotein na xivumbeko xa ring leyi heleleke, leswi landzelaka ku tirhisana kosungula na quinone xikan’we naku pfuriwa ka ndzhawu ya QB.
Muxaka wa II RC lowu kumekaka eka PSII complex ya tibakteriya to karhi ta phototrophic na cyanobacteria, algae na swimilani wu na nhlayiso wa xivumbeko na ntirho (38). Ku ringanisiwa ka xivumbeko loku kombisiweke eka Xifaniso xa 6 (D na E) ku kandziyisa ku fana exikarhi ka ti-RC ta PSII na ndhawu ya QB ya xivumbeko xa RC xa tibakteriya. Ku pimanisiwa loku i khale ku ri xikombiso xo dyondza maendlelo lama fambelanaka swinene ya ku boha na ku hunguta quinone. Minkandziyiso ya khale yi ringanyete leswaku ku cinca ka conformational ku fambisana na ku hunguteka ka PSII ka ti quinones ( 39 , 40 ). Hikokwalaho, loko hi tekela enhlokweni nhlayiso wa evolutionary wa RC, endlelo leri ro boha leri ari nga langutiwi khale ringa tlhela ri tirha eka ndzhawu ya QB ya PSII RC eka swimilani swa oxygenated phototrophic.
Rps ΔpufW (unlabeled pufW deletion) na PufW-His (C-terminal 10x His-tagged protein-W leyi kombisiweke kusuka eka ndzhawu ya ntumbuluko ya pufW) tinxaka. palustris CGA009 yi hlamuseriwile eka ntirho wa hina wa khale ( 16 ). Tinxaka leti na mutswari wa muxaka wa nhova wa isogenic swi vuyisiwile kusuka eka freezer hiku streaking nhlayo yintsongo ya ti cells eka PYE (yin’wana na yin’wana 5 g liter -1) (leyi hlayisiweke eka LB eka -80 °C, leyingana 50% ( w/v) glycerol) protein, yeast extract na succinate) agar [1.5% (w/v)] plate. Plate yi pfaleriwile vusiku hinkwabyo emunyameni eka mahiselo ya kamara ehansi ka swiyimo swa anaerobic, kutani yi voningiwa hi ku vonakala ko basa (~50 μmolm-2 s-1) loku nyikiwaka hi OSRAM 116-W halogen bulbs (RS Components, UK) kuringana 3 kuya eka 5 wa masiku kufikela loko ku humelela koloni yin’we. Koloni yin’we yi tirhisiwile ku chela 10 ml ya M22+ medium (41) leyi engeteriweke hi 0.1% (w/v) ya ti casamino acids (laha ku nga ta vuriwa M22). Ndhavuko wu byariwile ehansi ka swiyimo swa oxygen yale hansi emunyameni eka 34°C hiku rhurhumela hi 180 rpm kuringana 48 wa tiawara, kutani 70 ml ya culture yi cheriwile ehansi ka swiyimo leswi fanaka kuringana 24 wa tiawara. Semi-aerobic culture leyingana volume ya 1 ml yitirhisiwa ku chela 30 ml ya M22 medium eka 30 ml ya universal screw-top transparent glass bottle naswona yi cheriwa hi ku pfilunganyeka (~50μmolm-2 s-1) kuringana 48 wa tiawara hi sterile magnetic force Stirring rod. Kutani 30 ml ya culture yi cheriwile kwalomu ka 1 liter ya culture ehansi ka swiyimo leswi fanaka, leswi endzhaku switirhisiwile ku chela kwalomu ka 9 liters ya culture leyi voningeriweke eka ~200 μmolm-2 s-1 kuringana 72 wa tiawara. Ti cells ti tshoveriwile hi centrifugation eka 7132 RCF kuringana 30 wa timinete, titlhela ti suspended eka ~10 ml ya 20 mM tris-HCl (pH 8.0), naswona ti hlayisiwile eka -20°C kufikela loko swilaveka.
Endzhaku ko titimela, chela ti crystals tin’wana ta deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) na tiphilisi timbirhi ta Roche holoenzyme protease inhibitor (Merck, UK) eka ti cells letinga tlhela ti suspended. Eka 20,000 psi French pressure cell (Aminco, USA), ti cells ti kavanyetiwile minkarhi ya 8 kuya eka 12. Endzhaku ko susa ti cells letinga tshoveka na swilo leswinga khulukiki hiku centrifugation eka 18,500 RCF kuringana 15 wa timinete eka 4°C, membrane yi precipitated kusuka eka pigmented lysate hiku centrifugation eka 113,000 RCF kuringana 2 wa tiawara eka 43,000°C. Lahla xiphemu lexi n’okaka kutani u tlhela u suspend membrane ya muhlovo eka 100 kuya eka 200 ml ya 20 mM tris-HCl (pH 8.0) kutani u homogenize kufikela loko kungari na ti aggregates leti vonakaka. Membrane leyi yimisiweke yi pfaleriwile eka 20 mM tris-HCl (pH 8.0) (Anatrace, USA) leyingana 2% (w/v) β-DDM kuringana 1 wa awara emunyameni eka 4°C hiku hlanganisiwa hiku olova. Kutani centrifuge eka 70°C kuva yi n’oka 150,000 RCF eka 4°C kuringana 1 wa awara kuva ku susa leswinga sala leswinga khulukiki.
Membrane yo solubilizing kusuka eka muxaka wa ΔpufW yi cheriwile eka 50 ml ya DEAE Sepharose ion exchange column leyingana ti column volumes tinharhu (CV) ta binding buffer [20 mM tris-HCl (pH 8.0) leyingana 0.03% (w / v) β-DDM]. Hlantswa kholomo hi ti CV binding buffers timbirhi, kutani u hlamba kholomo hi ti binding buffers timbirhi letingana 50 mM NaCl. RC-LH116 complex yi hlantswiwile hi linear gradient ya 150 kuya eka 300 mM NaCl (eka binding buffer) eka 1.75 CV, naswona binding complex leyinga sala yi eluted hi binding buffer leyingana 300 mM NaCl eka 0.5 CV. Hlengeleta spectrum ya absorption exikarhi ka 250 na 1000 nm, hlayisa xiphemu lexi nga na absorbance ratio (A880/A280) leyikulu kutlula 1 eka 880 kuya eka 280 nm, yi dilute kambirhi eka binding buffer, naswona tirhisa endlelo leri fanaka nakambe eka kholomo ya DEAE On purification. Hlanganisa swiphemu leswingana ti ratios ta A880/A280 letinga e henhla kutlula 1.7 na ti ratios ta A880/A805 letinga e henhla kutlula 3.0, endla round ya vunharhu ya ion exchange, naswona hlayisa swiphemu leswingana ti ratios ta A880/A280 letinga e henhla kutlula 2.2 na ti ratios ta A880/A805 letinga ehenhla kutlula 5.0. Complex leyi basisiweke hi xiphemu yi hlanganisiwile kuya eka ~2 ml eka Amicon 100,000 molecular weight cut-off (MWCO) centrifugal filter (Merck, UK), naswona yi layichiwile eka Superdex 200 16/600 size exclusion column (GE Healthcare, US) leyingana 200 mM NaCl buffer, kutani yi hlantswiwa eka buffer leyi fanaka eka 1.5 CV. Hlengeleta ti absorption spectra ta size exclusion fraction, naswona hlanganisa ti absorption spectra letingana A880/A280 ratios eka 2.4 na A880/A805 ratios eka 5.8 kuya eka 100 A880, naswona hiku hatlisa u ti tirhisa eka ku lulamisela cryo-TEM grid kumbe ku hlayisa Hlayisa eka -80°C kufikela loko swilaveka.
Membrane yo solubilizing kusuka eka muxaka wa PufW-His yi cheriwile eka 20 ml ya HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) leyingana 200 mM NaCl na 0.03% (w/w)) eka IMAC buffer ( GE Healthcare). v) β-DDM ya xirhendzevutani]. Kholomo yi hlantswiwile hi ntlhanu wa ti CV ta IMAC buffer, kutani hi ntlhanu wa ti CV ta IMAC buffer letingana 10 mM histidine. Core complex yi eluted kusuka eka kholomo hi ntlhanu wa ti IMAC buffers letingana 100 mM histidine. Xiphemu lexi nga na RC-LH114-W complex xi hlanganisiwa kuya eka ~10 ml eka thangi leri hlanganisiweke leri hlomisiweke hi Amicon 100,000 MWCO filter (Merck, UK), ri cheriwa minkarhi ya 20 hi binding buffer, kutani ri cheriwa kuya eka 25 ml Eka kholomo ya DEAE Sepharose, mune wa ti CVs leti bohiweke eka buffer ti tirhisiwa ka ha ri emahlweni. Hlantswa kholomo hi mune wa ti CV binding buffers, kutani u hlantswa complex eka ti CV ta tsevu eka linear gradient ya 0 kuya eka 100 mM NaCl (eka binding buffer), na ti CV ta mune leti saleke letingana 100 mM binding buffer Ti complex leti saleke leti hlantswekeke eka sodium chloride leti hlanganisiweke na A880/A280 ratio yale henhla kutlula 2.4 na A880/A805 ratio yale henhla kutlula 4.6 wa swiphemu yi hlanganisiwile kuya eka ~2 ml eka Amicon 100,000 MWCO centrifugal filter, naswona yi tatiwe hi 1.5 CV IMAC ka ha ri emahlweni Buffer equilibrated Superdex 200 16/600 size exclusion column, kutani yi hlantswiwa eka buffer leyi fanaka eka 1.5 CV. Hlengeleta ti absorption spectra ta ti size-exclusion fractions kutani u hlanganisa ti absorption spectra letingana A880/A280 ratios eka 2.1 na A880/A805 ratios eka 4.6 kuya eka 100 A880, leswi hatlisaka switirhisiwa eka ku lulamisiwa ka frozen TEM grid kutani swi hlayisiwa eka -80°C kufikela loko swilaveka.
Leica EM GP immersion freezer yitirhisiwile ku lulamisa ti grids ta TEM ta mahiselo yale hansi. Complex yi cheriwile eka IMAC buffer kuya eka A880 ya 50, kutani 5μl yi layichiwile eka Glow-discharged leyintshwa ya QUANTIFOIL 1.2/1.3 carbon-coated copper mesh (Agar Scientific, UK). Hlayisa grid eka 20°C na 60% relative humidity kuringana 30 s, kutani u yi hlantswa yi oma kuringana 3 s, kutani u yi tima eka liquid ethane eka -176°C.
Data ya RC-LH114-W complex yi rhekhodiwile eka eBIC (Electronic Bioimaging Center) (British Diamond Light Source) hi microscope ya Titan Krios, leyi tirhaka eka voltage yo hatlisa ya 300kV, na ku kurisiwa ka vito ra 130,000× na eneji ya- Hlawula xivandla xa 20 eV. Gatan 968 GIF Quantum leyi nga na K2 peak detector yi tirhisiwile ku rhekhoda swifaniso eka movha wo hlayela ku hlengeleta datha. Vukulu bya pixel leyi kalibiweke i 1.048Å, naswona mpimo wa mpimo i 3.83 e-Å-2s-1. U hlengelete filimi hi tisekoni ta 11 ivi a yi avanyisa hi swiphemu swa 40. Tirhisa ndhawu leyi nga ni khaboni leswaku u tlhela u kongomisa microscope, ivi u hlengeleta tifilimi tinharhu eka khumbi rin’wana ni rin’wana. Hi ku angarhela, 3130 wa tifilimi ti hlengeletiwile, na mimpimo ya defocus exikarhi ka -1 na -3μm.
Data ya RC-LH116 complex yi hlengeletiwile hiku tirhisa microscope leyi fanaka e Asterbury Biostructure Laboratory (University of Leeds, UK). Data yi hlengeletiwile hi ndlela yo hlayela hi ku kurisiwa ka 130 k, naswona vukulu bya pixel byi kalibrate ku ya eka 1.065 Å hi mpimo wa 4.6 e-Å-2s-1. Muvi lowu wu rhekhodiwe hi tisekoni ta 12 naswona wu avanyisiwe hi swiphemu swa 48. Hiku angarhela, 3359 wa tifilimi ti hlengeletiwile, laha kungana mimpimo ya defocus exikarhi ka -1 na -3μm.
Ku lulamisiwa hinkwako ka datha ku endliwa eka phayiphi ya Relion 3.0 (42). Tirhisa Motioncorr 2 (43) ku lulamisa ku famba ka beam hi ku pima mpimo, kutani u tirhisa CTFFIND 4.1 (44) ku kumisisa parameter ya CTF (contrast transfer function). Ti photomicrographs leti tolovelekeke endzhaku ka switeji leswi swosungula swaku lulamisiwa ti kombisiwile eka Xifaniso 2. S16. Xifaniso xa ku hlawula ka otomatiki xi endliwa hi ku hlawula hi voko kwalomu ka 250 wa tipikseli ta 1000 wa swiphemuphemu eka furemu ya 250 wa tipikseli naswona ku hava ku hlawuriwa ka swikombo swa swiphemu swimbirhi (2D), hi ndlela yoleyo ku ala ku hlawuriwa koloko loku hlanganaka na ku thyakisa ka xikombiso kumbe ku nga ri na swihlawulekisi leswi vonakaka. Kutani, ku hlawuriwa ka otomatiki ku endliwile eka swifaniso hinkwaswo leswitsongo, naswona RC-LH114-W a yi ri ni swiphemuphemu swa 849 359, naswona RC-LH116 complex a yi ri ni swiphemuphemu swa 476 547. Ti particles hinkwato leti hlawuriweke ti hundze eka swirhendzevutani swimbirhi swa non-reference 2D classification, naswona endzhaku ka ku tsutsuma kun’wana na kun’wana, ti particles leti hlanganaka na ndzhawu ya carbon, ku thyakisa ka xikombiso, ku hava swihlawulekisi leswi nga erivaleni kumbe ti particles leti hlanganisaka swinene ta ariwa, leswi endlaka leswaku kuva na 772,033 (90.9%) na 359,678 (75.5%) ) Ti particles ti tirhisiwa eka 3D classification ya RC-LH114-W na RC-LH116 hi ku landzelelana. Modele wosungula wa 3D reference wu endliwile hiku tirhisa ndlela ya stochastic gradient descent. Hiku tirhisa modele wosungula tani hi xikombo, swiphemu leswi hlawuriweke swi avanyisiwile hi swiyenge swa mune eka 3D. Hiku tirhisa modele eka xiyenge lexi tani hi reference, endla 3D refining eka ti particles eka xiyenge lexikulu, kutani u tirhisa initial 15Å low-pass filter ku funengeta ndzhawu ya solvent, engetela 6 pixels ya soft edges, naswona post-process ti pixels ku lulamisa Gatan K2 peak Modulation transfer function ya top detector. Eka dataset ya RC-LH114-W, modele lowu wosungula wu cinciwile hiku susa density yo tiya eka makumu ya mask (yi hambanisiwile kusuka eka core complex density eka UCSF Chimera). Timodelo leti humelelaka (swiboho swa RC-LH114-W na RC-LH116 i 3.91 na 4.16 Å, hi ku landzelelana) swi tirhisiwa tanihi xikombo xa xirhendzevutani xa vumbirhi xa ku hlawuriwa ka 3D. Ti particles leti tirhisiweke ti hlengeletiwile eka ntlawa wosungula wa 3D naswona ati na vuxaka byo tiya na vaakelani. Ku hlangana kumbe ku pfumaleka ka swihlawulekisi swa xivumbeko leswi nga erivaleni. Endzhaku ka xirhendzevutani xa vumbirhi xa ku hlawuriwa ka 3D, xiyenge lexi nga na xiboho xa le henhla xi hlawuriwile [Eka RC-LH114-W, xiyenge xin’we i 377,703 wa swiphemuphemu (44.5%), eka RC-LH116, ku na swiyenge swimbirhi, leswi hlanganisaka 260,752 wa swiphemuphemu (54.7%) , Laha swi fanaka ntsena loko swi ringanisiwile endzhaku ka ku rhendzeleka ko sungula hi ku hambana kutsongo]. Ti particles leti hlawuriweke titlhela ti humesiwa eka bokisi ra 400-pixel naswona ti antswisiwa hiku antswisiwa ka 3D. Xisirhelelo xa solvent xi endliwa hiku tirhisa 15Å low-pass filter yosungula, 3 pixel map expansion na 3 pixel soft mask. Hiku tirhisa ku antswisiwa ka CTF hi xiphemu xin’wana na xin’wana, ku lulamisiwa ka ku famba ka xiphemu xin’wana na xin’wana na xirhendzevutani xa vumbirhi xa ku antswisiwa ka CTF hi xiphemu xin’wana na xin’wana, ku antswisiwa ka 3D, ku funengetiwa ka solvent na ku lulamisiwa ka le ndzhaku swi endliwa endzhaku ka goza rin’wana na rin’wana ku ya emahlweni ku antswisiwa xivumbeko lexi humelelaka. Hi ku tirhisa nhlayo ya ku tsema ya FSC (Fourier Shell Correlation Coefficient) ya 0.143, swiboho swa timodeli to hetelela ta RC-LH114-W na RC-LH116 i 2.65 na 2.80Å, hi ku landzelelana. FSC curve ya modele yo hetelela yi kombisiwile eka Xifaniso 2. S17.
Ntlhandlamano hinkwawo wa tiphrotheyini wu dawunilodiwile eka UniProtKB: LH1-β (PufB; UniProt ID: Q6N9L5); LH1-α (PufA; ID ya UniProt: Q6N9L4); RC-L (PufL; ID ya UniProt: O83005); RC-M (PufM; ID ya UniProt: A0A4Z7); RC-H (PuhA; ID ya UniProt: A0A4Z9); Tiphrotheyini-W (PufW; ID ya UniProt: Q6N1K3). SWISS-MODEL (45) yi tirhisiwile ku aka modele wa homology wa RC, lowu nga na ntlhandlamano wa ti protein ta RC-L, RC-M na RC-H na xivumbeko xa crystal xa Rba. sphaeroides RC yi tirhisiwile tanihi xifaniso (PDB ID: 5LSE) (46). Tirhisa xitirhisiwa xa “fit map” eka UCSF Chimera ku ringanisa modele lowu endliweke eka mepe (47), ku antswisa xivumbeko xa tiphrotheyini, na cofactor [4×BChl a (vito ra masalela ya layiburari ya monomer = BCL), 2×BPh a (BPH), muxaka wun’we kumbe timbirhi ta UQ10 (U10), nsimbi yin’we leyi nga riki ya heme (Fe) na yin’we ya 3,4-dihydrohexacarbonylcholine (QAK)] tirhisa Coot (48) ku engetela. Leswi QAK yi nga kumekiki eka layiburari ya monomer, yi parameterized hi ku tirhisa xitirhisiwa xa eLBOW eka PHENIX ( 49 ).
Endzhaku ka sweswo, ku akiwile xiyenge lexitsongo xa LH1. Eku sunguleni, xitirhisiwa xo aka xa otomatiki eka PHENIX (49) xi tirhisiwile ku aka hi ku tisungulela xiphemu xa ntlhandlamano wa LH1 hi ku tirhisa mepe na ntlhandlamano wa tiphrotheyini ta LH1-α na LH1-β tanihi ku nghenisa. Hlawula subunit ya LH1 leyi heleleke swinene, yi humesa u yi layicha eka Coot, chela hi voko ntlhandlamano lowu kayivelaka eka yona, naswona hi voko u antswisa xivumbeko hinkwaxo loko unga se chela ti BCls timbirhi a (BCL) na spirilloxanthin (CRT) [kuya hi ti Rps leti faneleke Ku tsindziyela ka LH1 complex xikan’we na nhlayo ya carotenoid leyi tivekaka. Tinxaka ta swiharhi (17)]. Kopa subunit ya LH1 leyi heleleke, naswona tirhisa UCSF Chimera “Docking Map Tool” ku dock eka ndzhawu leyi nga ekusuhi leyi nga riki model ya LH1 density, kutani u yi antswisa eka Coot; phindha endlelo ku kondza ti subunits hinkwato ta LH1 ti modeliwa. Eka xivumbeko xa RC-LH114-W, hiku humesa density leyinga averiwangiki eka Coot, protein yi avanyisiwile kusuka eka swiphemu leswinga sala leswingariki ti protein eka mepe wa USCF Chimera naswona xitirhisiwa xa Autobuild xitirhisiwa ku simeka modele wosungula, na ti subunits letinga sala (protein-W) Modeling. Eka PHENIX (49). Engetelani ntlhandlamano wihi na wihi lowu kayivelaka eka modele lowu humelelaka eka Coot (48), ivi u antswisa hi voko subunit hinkwayo. Density leyi saleke leyi nga averiwangiki yi fambelana na nhlanganelo wa ti lipids (PDB monomer library ID ya CDL = CDL, POPC = 6PL na POPG = PGT), β-DDM detergent (LMT) na ti molecules ta UQ10 (U10). Tirhisa PHENIX optimization (49) na manual optimization eka Coot (48) ku hetisisa modele wosungula lowu heleleke kufikela loko tinhlayo ta modele na khwalithi ya mahlo ya fit swinga antswisiwa kuya emahlweni. Eku heteleleni, tirhisa LocScale (50) ku olovisa mepe wa ndhawu, ivi u endla swirhendzevutani swin’wana swo hlayanyana swa ku modela density leyi nga averiwangiki na ku antswisiwa ka otomatiki na hi voko.
Ti peptides hi ku hambana ka tona, ti cofactors na ti lipids tin’wana na ti quinones leti docked endzeni ka ti densities ta tona hi ku hambana ka tona ti kombisiwile eka Swifaniso 1 na 2. S18 kuya eka S23. Vuxokoxoko bya tinhlayo bya modele wo hetelela byi kombisiwile eka Tafula ra S1.
Handle ka loko swi boxiwile hi ndlela yin’wana, ti UV/Vis/NIR absorption spectra ti hlengeletiwile eka Cary60 spectrophotometer (Agilent, USA) eka 1 nm intervals kusuka eka 250 nm kuya eka 1000 nm na nkarhi wo hlanganisa wa 0.1s.
Hlanganisa xikombiso eka quartz cuvette leyingana ndlela ya 2 mm kuya eka A880 ya 1, naswona hlengeleta absorption spectrum exikarhi ka 400 na 1000 nm. Ti circular dichroic spectra ti hlengeletiwile eka Jasco 810 spectropolarimeter (Jasco, Japan) eka 1 nm intervals exikarhi ka 400 nm na 950 nm eka scan rate ya 20 nm min-1.
Molar extinction coefficient yikumiwa hiku chela core complex kuya eka A880 ya kwalomu ka 50. Hlanganisa 10μl volume eka 990μl binding buffer kumbe methanol, naswona hlengeleta absorption spectrum hiku hatlisa ku hunguta ku onhaka ka BChl. Nhlayo ya BChl ya xikombiso xin’wana na xin’wana xa methanol yi hlayiwile hi extinction coefficient eka 771 nm ya 54.8 mM-1 cm-1, naswona extinction coefficient yikumiwile ( 51 ). Avanyisa nhlayo ya BChl leyi pimiweke hi 32 (RC-LH114-W) kumbe 36 (RC-LH116) ku kumisisa nhlayo ya core complex, leyi endzhaku yi tirhisiwaka ku kumisisa spectrum ya absorption ya xikombiso lexi fanaka lexi hlengeletiweke eka buffer Extinction coefficient. lavana. Mipimo yinharhu leyi phindha-phindhiweke yi tekiwile eka xikombiso xin’wana na xin’wana, naswona avhareji ya absorbance ya BChl Qy maximum yitirhisiwile eka xibalo. Nhlayo ya ku herisiwa ka RC-LH114-W leyi pimiweke eka 878 nm i 3280±140 mM-1 cm-1, kasi nhlayo ya ku herisiwa ka RC-LH116 leyi pimiweke eka 880 nm i 3800±30 mM-1 cm-1.
UQ10 yi pimiwile hi ku ya hi ndlela leyi nga eka (52). Hiku komisa, reverse phase HPLC (RP-HPLC) yi endliwile hiku tirhisa Agilent 1200 HPLC system. N’okisa kwalomu ka 0.02 nmol ya RC-LH116 kumbe RC-LH114-W eka 50μl ya 50:50 methanol:chloroform leyi nga na 0.02% (w/v) ferric chloride, naswona chela Beckman Coulter Ultrasphere ODS 4.6 mm leyi ringaniseriweke ka ha ri emahlweni yi n’okisela eka 1 ml-1 min-1 eka 40°C eka HPLC xitofu (80:20 methanol:2-propanol) eka kholomo ya ×25 cm. Endla isocratic elution eka HPLC solvent ku langutisisa absorbance eka 275 nm (UQ10), 450 nm (carotenoids) na 780 nm (BChl) kuringana 1 wa awara. Nhlohlorhi eka 275 nm chromatogram eka 25.5 wa timinete yi hlanganisiwile, leyi ayi nga ri na swihlanganisi swin’wana leswi voniwaka. Ndzhawu leyi hlanganisiweke yitirhisiwa ku hlayela nhlayo ya molar ya UQ10 leyi humesiweke hiku kongomisa eka calibration curve leyi hlayiweke kusuka eka injection ya pure standards kusuka eka 0 kuya eka 5.8 nmol (Figure S14). Xikombiso xin’wana na xin’wana xi kamberiwile hi ku phindha-phindha kanharhu, naswona xihoxo lexi vikiweke xi fambelana na SD ya avhareji.
Nkatsakanyo lowungana RC-LH1 complex lowungana Qy absorption yale henhla ya 0.1 wu lulamisiwile hi 30 μM leyi hungutiweke ya horse heart cytochrome c2 (Merck, UK) na 0 kuya eka 50 μMUQ2 (Merck, UK). Tisampulu tinharhu ta 1-ml ti lulamisiwile eka nhlayo yin’wana na yin’wana ya UQ2 naswona ti pfaleriwile vusiku hinkwabyo emunyameni eka 4°C ku tiyisisa ku tolovela hiku hetiseka eka munyama loko kungase pimiwa. Nkatsakanyo lowu wu layichiwile eka OLIS RSM1000 modular spectrophotometer leyi hlomisiweke hi 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm middle na 0.6 mm outlet slits. Sefo ya 600 nm long pass yi vekiwa eka nyangwa wa phototube ya xikombiso na chubu ra reference photomultiplier ku susa ku vonakala ka excitation. Absorbance yi langutisiwile eka 550 nm hi nkarhi wo hlanganisa wa 0.15 s. Ku vonakala ka excitation ku humesiwa kusuka eka 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) hiku tirhisa fibre optic cable hi 90% wa matimba hiku tirhisa DC2200 controller (Thorlabs Ltd., UK) naswona yi humesiwa eka xihlovo xa ku vonakala hi angle ya 90° ya. Beam yo pima yi lwisana na xivoni ku vuyisa ku vonakala kwihi na kwihi loku eku sunguleni a ku nga ngheniwi hi xikombiso. Languta absorbance 10 s loko kungase voningi 50 s. Kutani absorbance yitlhele yi vekiwa tihlo kuringana 60 s emunyameni ku kambela mpimo lowu quinolol yi hungutaka cytochrome c23 + hiku tisungulela (vona Figure S8 for raw data).
Data yi lulamisiwile hiku ringanisa linear initial rate eka 0.5 kuya eka 10 s (kuya hi UQ2 concentration) xikan’we naku averisa ti rates ta tisampulu hinkwato tinharhu eka concentration yin’wana na yin’wana ya UQ2. Nhlayo ya RC-LH1 leyi hlayiweke hi extinction coefficient leyi faneleke yitirhisiwile ku hundzula mpimo kuya eka catalytic efficiency, leyi plotiweke eka Origin Pro 2019 (OriginLab, USA), naswona yi ringanisiwile eka modele ya Michaelis-Menten ku kumisisa mimpimo leyi vonakaka ya Km na Kcat.
Eka mimpimo ya ku amukeriwa ka nkarhinyana, xikombiso xa RC-LH1 xi cheriwile kuya eka ~2μM eka IMAC buffer leyingana 50 mM sodium ascorbate (Merck, USA) na 0.4 mM Terbutin (Merck, USA). Ascorbic acid yitirhisiwa tani hi sacrificial electron donor, naswona tert-butaclofen yitirhisiwa tani hi QB inhibitor kutiyisisa leswaku RC donor leyikulu yitshama yirikarhi yihunguteka (leswivulaka leswaku, kungari photooxidized) eka maendlelo hinkwawo ya ku pima. Kwalomu ka 3 ml ya xikombiso yi cheriwa eka sele leyi rhendzelekaka ya ntolovelo (kwalomu ka 0.1 m hi vukulu, 350 RPM) leyingana 2 mm ya ku leha ka ndlela ya optical kutiyisisa leswaku xikombiso lexinga eka ndlela ya laser xina nkarhi lowu ringaneleke wa ku cinca cinca ka munyama exikarhi ka ti excitation pulses. Tirhisa ~100-fs laser pulses ku kurisa Ti: Sapphire laser system (Spectra Physics, USA) ku hlohlotela xikombiso eka 880 nm eka repetition rate ya 1 kHz (20 nJ eka NIR kumbe 100 nJ eka Vis). Loko unga se hlengeleta data, veka xikombiso eka ku vonakala ka excitation kuringana kwalomu ka 30 wa timinete. Ku hlangana na swilo swita endla leswaku QA yinga tirhi (swinga endleka ku hunguta QA kan’we kumbe kambirhi). Kambe hi kombela u xiya leswaku endlelo leri ra hundzuka hikuva endzhaku ka nkarhi wo leha wa ku cinca ka munyama, RC yi ta tlhelela hakatsongotsongo eka ntirho wa QA. Helios spectrometer (Ultrafast Systems, USA) yitirhisiwile ku pima ti spectra ta nkarhinyana hi nkarhi wo hlwela wa -10 kuya eka 7000 ps . Tirhisa software ya Surface Xplorer (Ultrafast Systems, USA) ku susa mintlawa ya tisethi ta data, kutani u hlanganisa na ku ringanisa. Tirhisa phakeji ya software ya CarpetView (Light Conversion Ltd., Lithuania) ku tirhisa sete ya datha leyi hlanganisiweke ku kuma ti spectra to hambana leti fambelanaka na ku bola, kutani u tirhisa ntirho lowu hlanganisaka swikombiso swo tala na nhlamulo ya xitirhisiwa ku fambelana na ku hundzuka ka spectral ya wavelength yin’we eka Origin (OriginLab, USA).
Hilaha swi boxiweke hakona laha henhla (53), filimi ya photosynthetic leyi nga na LH1 complex leyi pfumalaka RC na peripheral LH2 antenna yi lulamisiwile. Membrane yi cheriwile eka 20 mM tris (pH 8.0) kutani yi layichiwile eka quartz cuvette leyingana 2 mm optical path. 30nJ laser pulse yitirhisiwile ku hlohlotela xikombiso eka 540 nm hi nkarhi wo hlwela wa -10 kuya eka 7000 ps. Endla sete ya data tanihilaha swi hlamuseriweke hakona eka Rps. pal xikombiso.
Membrane yi pelleted hi centrifugation eka 150,000 RCF kuringana 2 wa tiawara eka 4°C, kutani absorbance ya yona eka 880 nm yitlhele yi suspended eka 20 mM tris-HCl (pH 8.0) na 200 mM NaCl. N’okisa membrane hiku hlanganisa hiku nonoka eka 2% (w/v) β-DDM kuringana 1 wa awara emunyameni eka 4°C. Xikombiso xi cheriwile eka 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) kuya eka nhlayo ya ti protein ta 2.5 mg ml-1 (Bio-Rad analysis). Ku lulamisiwa kun’wana ku endliwile kusuka eka ndlela leyi kandziyisiweke khale (54), kusukela hiku cheriwa ka 50 μg protein kuya eka ntsengo wa 50 μl TEAB leyingana 1% (w/v) sodium laurate (Merck, UK). Endzhaku ka sonication kuringana 60 s, yi hungutiwile hi 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) eka 37°C kuringana 30 wa timinete. Eka S-alkylation, pfalela xikombiso na 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) kutani u chela kusuka eka 200 mM isopropanol stock solution kuringana 10 wa timinete eka mahiselo ya kamara. Ku gayeriwa ka proteolytic swiendliwile hiku cheriwa 2 μg ya trypsin/endoproteinase Lys-C mixture (Promega UK) naswona swi pfaleriwile eka 37°C kuringana 3 wa tiawara. Laurate surfactant yi humesiwile hiku cheriwa 50 μl ethyl acetate na 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) xikan’we na vortexing kuringana 60 s. Ku hambanisiwa ka phase swi hlohleteriwile hi centrifugation eka 15,700 RCF kuringana 5 wa timinete. Kuya hi protocol ya muendli, C18 spin column (Thermo Fisher Scientific, UK) yitirhisiwile ku aspirate hi vukheta naku susa munyu eka xiphemu xale hansi lexinga na peptide. Endzhaku ko omisiwa hi vacuum centrifugation, xikombiso xi pfanganisiwile na 0.5% TFA na 3% acetonitrile, naswona 500 ng yi kamberiwile hi nanoflow RP chromatography leyi hlanganisiweke na mass spectrometry hiku tirhisa ti parameters ta sisiteme leti hlamuseriweke hi vuxokoxoko khale.
Tirhisa MaxQuant v.1.5.3.30 (56) eka ku tiveka ka ti protein na ku pima nhlayo ku lavisisa Rps. database ya proteome ya palustris (www.uniprot.org/proteomes/UP000001426). Data ya mass spectrometry proteomics yi vekiwile eka ProteomeXchange Alliance hi ku tirhisa vuhlayiselo bya mutirhisani wa PRIDE (http://proteomecentral.proteomexchange.org) ehansi ka xihlawulekisi xa dataset PXD020402.
Ku kamberiwa hi RPLC leyi hlanganisiweke na electrospray ionization mass spectrometry, RC-LH1 complex yi lulamisiwile kusuka eka wild-type Rps. Hi ku tirhisa ndlela leyi kandziyisiweke khale (16), nhlayo ya tiphrotheyini leyi humesiweke eka tisele ta palustris a yi ri 2 mg ml-1 eka 20 mM Hepes (pH 7.8), 100 mM NaCl na 0.03% (w/v) β- (Nxopaxopo wa Bio-Rad) ) DDM. Kuya hi protocol ya muendli, tirhisa 2D purification kit (GE Healthcare, USA) ku humesa 10 μg ya ti protein hi ndlela ya precipitation, naswona u n’okisa precipitate eka 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile na 20% (v/v) mati. Ntlhanu wa ti microliters ti kamberiwile hi RPLC (Dionex RSLC) leyi hlanganisiweke na mass spectrometry (Maxis UHR-TOF, Bruker). Tirhisa kholomo ya MabPac 1.2×100 mm (Thermo Fisher Scientific, UK) ku hambanyisa eka 60°C na 100μlmin -1, na gradient ya 85% (v / v) solvent A [0.1% (v / v) FA na 0.02% (V/v) TFA aqueous solution] kuya eka 85%(v/v) solvent B [0.1%(v/v) FA na 0.02%(v/v) eka 90%(v/v) acetonitrile TFA] Hiku tirhisa xihlovo xa ionization xa electrospray xa ntolovelo na ti parameter ta default kutlula 60 wa timinete, mass spectrometer yikuma 100 kuya eka 2750 m/z (mass-to-charge ratio). Hi ku pfuniwa hi xitirhisiwa xa ExPASy bioinformatics resource portal FindPept (https://web.expasy.org/findpept/), mepe spectrum ya vukulu eka ti subunits ta complex.
Ti cells ti kurisiwile kuringana 72 wa tiawara ehansi ka 100 ml ya NF-low (10μMm-2 s-1), medium (30μMm-2 s-1) kumbe high (300μMm-2 s-1) light. M22 medium (M22 medium laha ammonium sulfate yi tshikiweke naswona sodium succinate yi siviwa hi sodium acetate) eka bodlhela ra 100 ml ra screw-top (23). Eka ntlhanu wa swirhendzevutani swa 30-s, vuhlalu bya nghilazi bya 0.1 micron byi endliwe vuhlalu eka volume ratio ya 1:1 ku lyse ti cells naswona byi titimela eka ayisi kuringana 5 wa timinete. Mhaka leyinga khulukiki, ti cells letinga tshovekangiki na vuhlalu bya nghilazi swi susiwile hi centrifugation eka 16,000 RCF kuringana 10 wa timinete eka benchtop microcentrifuge. Membrane yi hambanisiwile eka Ti 70.1 rotor na 100,000 RCF eka 20 mM tris-HCl (pH 8.0) na 40/15% (w/w) sucrose gradient kuringana 10 wa tiawara.
Hilaha swi hlamuseriweke hakona eka ntirho wa hina lowu hundzeke, immunodetection ya His tag eka PufW (16). Hiku komisa, purified core complex (11.8 nM) kumbe membrane leyingana nhlayo leyi fanaka ya RC (leyi kumiwaka hiku oxidation ku susa spectrum ya ku hambana leyi hungutiweke xikan’we naku fambelana na ndzhwalo eka gel leyi tshuburiweke) eka 2x SDS loading buffer (Merck, UK) leyi cheriweke kambirhi. Ti proteins ti hambanisiwile eka replica 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). Gel yi cheriwe Coomassie Brilliant Blue (Bio-Rad, UK) ku layicha naku vona hi mahlo ya mianakanyo subunit ya RC-L. Protein eka gel ya vumbirhi yi hundziseriwe eka methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) kuva yi endliwa immunoassay. Membrane ya PVDF yi pfaleriwile eka 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 na 5% (w / v) skimmed milk powder, kutani yi pfaleriwa na anti-His primary antibody (eka Dilute the antibody buffer [50 mM tris-HCl (pH 7.6), kutani yi pfaleriwa na antibody buffer [50 mM tris-HCl (pH 7.6), 150 mM NaCl na 0.05% (v/v) Tween-20] eka 1:1000 A190-114A, Bethyl Laboratories, USA) kuringana 4 wa tiawara. Endzhaku ko hlamba minkarhi ya 3 kuringana 5 wa timinete eka antibody buffer, membrane yi hlanganisiwile na horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (diluted 1:10,000 eka antibody buffer) Yi pfaleriwa kuva yi pfumelela ku kumiwa (5 wa timinete endzhaku ka 3 wa ku hlantswiwa eka antibody buffer) hiku tirhisa WESTAR ETA C 2.0 chemiluminescence substrate (Cyanagen, Italy) na Amersham Imager 600 (GE Healthcare, UK).
Hi ku dirowa ku hangalasiwa ka matimba ya gel yin’wana na yin’wana leyi nga na swivati ​​kumbe layini ya immunoassay, ku hlanganisa ndhawu leyi nga ehansi ka nhlohlorhi na ku hlayela mpimo wa matimba ya RC-L (gel leyi nga na swivati) na Protein-W (immunoassay), eka ImageJ (57) Endla xifaniso. Ti ratios leti ti hundzuriwile kuya eka ti molar ratios hiku ehleketa leswaku ratio ya RC-L kuya eka protein-W eka xikombiso xa pure RC-LH114-W akuri 1:1 naku normalisa sete hinkwayo ya data kuya hi sweswo.
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Xivutiso lexi xi tirhisiwa ku kambela loko u ri muendzi na ku sivela ku rhumeriwa ka swipamu hi ku tisungulela.
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
Xivumbeko xa high-resolution xa light trap 1 complex eka reaction center xinyika vutivi byintshwa eka quinone dynamics.
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
Xivumbeko xa high-resolution xa light trap 1 complex eka reaction center xinyika vutivi byintshwa eka quinone dynamics.
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