Lino†Kkeyala yamazubaano: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Busena bwakuyobolola mumuni 1 (RC-LH1) ncecibeela cipati cakupanga mumuni wamabacteria aajisi mumuni uusalala pyu. Twakatalisya zibeela zyobile zya cryo-electron microscopy zya RC-LH1 complex kuzwa ku Rhodopseudomonas palustris. I 2.65-Å resolution structure ya RC-LH114-W complex ilijisi 14 subunit LH1 loops izingulukide RC, eeyo iinyonganizyigwa a protein W, mpawo complex iitakwe protein-W ilijisi RC composition iizingulukidwe a RC. Kujala 16 subunit LH1 loop. Kweezyanisya zibeela eezi kupa kumvwisisisya kuchincha kwa quinone mu RC-LH1 complex, kubikkilizya akusanduka kwakaindi kutazyibidwe aawo nokwakali kujatikizyigwa quinone kubusena bwa RC QB, antoomwe abusena bwama quinone aajatikizyidwe, alo aagwasya kuti apegwe ku RC. Bupange bulibedelede bwa W protein bulesya kujalwa kwa LH1 loop, aboobo kupa kuti kube nzila yakufwambaanya kuchincha kwa quinone/quinolone.
Nguzu zizwa ku photosynthesis zilakonzya kubamba buumi boonse anyika, alimwi zilakonzya kugwasya kapati mukubelesya zuba. Nikuba kuti zisumpula nzila yakupanga zuba munyika yoonse, tuzunda twamumuni tutondeezya nguzu zyaandeene-andeene anguzu zyakusanduka kwazyakulya. Zilakonzya kutantamuka kupanga mumuni mpawo zikomene mbuli mabakiteriya aatakwe busani mumudima, zilakonzya kubamba nitrogen a carbon dioxide, kupanga haayidirojeni, akunyonganya zintu zijisi tununkilizyo (1-3). Kuti kube manguzu kuzintu eezyi, mumuni weelede kusanduka chakufwambaana mpawo ube manguzu aamakemikalu. Eeyi nzila italika aawo antenna iijata mumuni noitambula mumuni mpawo itola manguzu aajatikizyidwe kubusena bwakuchitila zintu (RC), aboobo kutalika kwaandaana kwanguzu (4 – 7). Chibeela chakusanguna chakupanga mumuni mumabacteria aajisi mumuni uusalala pyu chipangidwe amusyobo wa 2 RC, uuzingulukidwe a light-harvesting complex 1 (LH1), mpawo upanga RC-LH1 core complex. LH1 yakapangwa amulongo wama αβ heterodimers, aumwi ulajata ma molecule obile aa bacterial chlorophyll (BChl) a carotenoids yomwe naa zyobile (8-12). I LH1 antenna nguba ilijisi 16 naa 17 αβ heterodimers izingulukide RC (9-13) munzila iijalidwe, pesi muli zimwi zibeela, ma peptide aazwa mumubili alalesya kuzumanana kwa LH1, aboobo asumpula kumwaika kwa quinol/quinone aakati ka RC a cytochrome bc complex (1111, 13). Musamu uuzwa mumuni uutegwa Rhodopseudomonas (Rps.) nchintu chikonzya kumvwisisisya manguzu akusanduka kwamagesi aagwasya kupanga zuba. Bupange bwakusaanguna bwa Rps. Ikozyanisyo ya palustris RC-LH1 complex ni RC, izingulukidwe a 15 heterodimeric LH1 loops, eezyo zinyonganizyigwa a protein iitazyibidwe yiitwa kuti "Protein W" (14). Ma protein-W akazyibwa kuti RPA4402, eeyo iili 10.5kDa ipuloteni iitazyibidwe iijisi zibeela zyotatwe zya transmembrane helices (TMH) (16). Tulayeeya kuti tuchinche zina lya rpa4402 gene iibamba ma protein W kuti ibe pufW kuti yeendelane amazina aabelesyegwa kuma genes aajisi RC-L, M (pufL, pufM) antoomwe a LH1α, β (pufA, pufB) subunits. Chakugambya, ma protein-W ali buyo mu 10% ya RC-LH1, kutondeezya Rps. palustris ilazyala RC-LH1 zyobile ziindene. Aawa, twaambilizya zibeela zya cryo-EM (cryo-EM) ziboneka kabotu zyazibeela zyobile, chimwi chili ama protein W a 14 αβ heterodimers, chimwi chitakwe ma protein W antoomwe a 16 Heterodimer LH1 loop. Bupange bwesu butondezya kucinca kwantaamu mukumvwisisisya RC-LH1 complex ya Rps. palustris, nkaambo twakavwuntauzya mweelwe uukozyenye wamusyobo umwi awumwi mpawo twakajisi bupanduluzi buzulide bwakusala kabotu-kabotu peptide imwi aimwi alimwi amabala aajatikizyidwe alimwi amafuta aajatikizyidwe alimwi ama quinones. Kweezyanisya zibeela eezi kutondeezya kuti ma protein otatwe aa TMH-W aatajanwi muli RC-LH1 kusikila lino apanga nzila ya quinone kuti ifwambaane kucincanya quinone/quinolone. Mweelwe wamasena aajatikizyidwe aamafuta a quinone akajanwa, mpawo twakayubununa kuchincha kupya kwabupange nikwakamana kusanganya quinone a RC, ooko kukonzya kweelela ku photosystem II (PSII) RC ya oxygenated phototrophic organisms. Nzyotujana zipa muzeezo mupya kujatikizya kweenda kwa quinone/quinolone kuswaangana akucincanya mu RC-LH1 core complex ya purple phototrophic bacteria.
Kuti kube chiiyo chilamfu chazibeela zyobile zijanika mu Rps. palustris, twaandaanya RC-LH1 imwi aimwi kwiinda mukubelesya nzila zya biochemical. I protein W-deficient complex (aawa yiitwa kuti ΔpufW) yakasalazyigwa kuzwa kumusyobo uutakwe pufW gene (16), mpawo buyo RC-LH1 complex yomwe ilakonzya kupangwa. Ipuloteni W-ijisi chikozyano chipangwa amusyobo. Ipuloteni W yamusyobo ooyu yakacincwa a 10x His tag kumamanino aa C, kuchitila kuti ipuloteni W-ijisi kkompyuta ikonzye kusanganyigwa kabotu amapulotiini W aatakwe kwiinda mukulesya nsimbi. Ibbazu lyaandaanizyidwe kabotu (16) Affinity Chromatography (IMAC).
Mbuli mbokutondezyedwe achifanikisyo 1, zyoonse zyobile zilijisi zibeela zyotatwe zya RC (RC-L, RC-M a RC-H) zizingulukidwe a LH1 antenna. Bupange bwa 2.80-A bwacibeela citakwe ma protein-W butondezya 16 αβ heterodimers, zipanga LH1 loop iijalidwe iizingulukide RC, kuzwa lino yiitwa kuti RC-LH116 complex. Bupange bwa 2.65Å bwa ma protein-W-ajisi 14-heterodimer LH1 yakanyonganizyigwa a protein-W, lino yiitwa kuti RC-LH114-W.
(A a B) Chitondeezyo chaatala cha musamu. (C a D) Mibala iiswaangene itondezyedwe mumisamu. (E a F) Zibeela zyakabonwa kuzwa atala aa cytoplasmic zili ama peptides a LH1 subunits zitondezyedwe muma katuni, mpo zililembedwe nsamu kuzwa ku protein-W gap [kweendelana anamba ya Rba. sphaeroides complex (13)]. Kuli LH1-α, mubala wacibeela chamapulotini ngusalala pyu; kuli LH1-β, mubala wacibeela ca ma protein ngu bbuluu; kuli ma protein-W, ma protein alisalala pyu; kuli RC-H, nchintu chiyumu; kuli RC-L, ngu Orange; ya RC-M, magenta. Ma cofactors aimininwa amisamu, mubala wa sokwe wiiminina ma molecule aa BChl antoomwe a BPh a, mubala wa sokwe wiiminina ma molecule aa carotenoids, mpawo mubala wa sokwe wiiminina ma molecule aa UQ10. (G a H) Kuyungizya kubona kwa protein-W gap mubusena bweelene a RC-LH114-W complex (G) antoomwe a RC-LH116 complex (H). Ma cofactors atondezyegwa munzila yakuzuzya busena, chelated quinone itondezyegwa mubala wa lubululu. Mulyango wa protein-W utondezyedwe amuzila wa bbuluu mu (G), mpawo tuzila tusyoonto oomo quinone/quinolol mpozimwaika a LH116 ring zitondezyedwe amuzila uusiya mu (H).
Cifwanikiso 1 (A a B) chitondeezya RC kazingulukidwe aama array aajulikide naa aajalidwe aa LH1αβ heterodimers, aumwi wazyo uujatikizya BChl zyobile a carotenoid yomwe (Cifwanikiso 1, C a D). ziiyo zyakachitika musule zyakatondezya kuti Rps ninzila ya LH1. Munzila yakupanga zintu zipona zya spirulina xanthin, misyobo eeyi ilijisi misyobo ya carotenoids (17). Nokuba boobo, spiropyrroxanthin ngo carotenoid mupati mpawo bunji bwawo buli kabotu. Aboobo, twakasala kubelesya spiroxanthin kumasena woonse aa LH1. Ma polypeptide aa alpha antoomwe a beta nga TMH yomwe iijisi zibeela zyaanze zifwiifwi (Chitondeezyo 1, A, B, E, a F). Nikuba kuti bunji bwazibeela zili kkumi amusanu azibili ku C-terminus tiibwakabonwa pe, alpha polypeptide yakaandaanizyigwa kuzwa ku Met1 kusika ku Ala46 muzibeela zyoonse zyobile. β polypeptide yakagwisigwa kuzwa ku Gly4 kuya ku Tyr52 mu RC-LH116, mpawo kuzwa ku Ser5 kuya ku Tyr52 mu RC-LH114-W. Taakwe bunji bwa 3 naa 4 N-terminal naa 13 C-terminal zyakasyeede zyakabonwa (Chitondeezyo S1). Kuvwuntauzya kwa mass spectrometry kwa RC-LH1 complex yakasanganizigwa kuzwa ku wild-type strain kwakatondezya kuti busena bwakasweekede bwakazwa kukutyompokelwa kwama peptide aaya (Chitondeezyo S1 a S2). Kubamba kwa N-kumamanino kwa α-Met1 akwalo kwakabonwa (f). Kuvwuntauzya kwakatondezya kuti α-peptide ilijisi zibeela zya fMet1 kusikila ku Asp42/Ala46/Ala47/Ala50, mpawo β-peptide ilijisi zibeela zya Ser2 kusikila ku Ala53, eezyo zileendelana a low-temperature EM density map.
Kuswaangana kwa α-His29 a β-His36 kupa kuti BChls kabalanga abusyu; αβ heterodimer imwi aimwi ilabungana abasimukobonyina kuti ipange ntambo iijalukide (RC-LH114-W) naa ntalisyo iijalikide (RC-LH116) kuzinguluka RC The exciton coupled pigment array (Figure 1, C a D). Kweezyanisya a 877 nm band ya RC-LH114-W, 880 nm absorption red shift ya RC-LH116 3 nm (Chitondeezyo 2A). Nokuba boobo, nzila ya circular dichroism ilikozyene (Chitondeezyo 2B), kutondezya kuti nikuba kuti kuli kwiindana aakati kazyintu zijulidwe azijalidwe, busena bwa BChls bulikozyene. Kusanduka kwa redshift kwakuyoya kulakonzya kuba akaambo kakucheya kwakuzungaana kwakupya akuyungizya kukkalwa moyo aamulongo uujalilidwe (18, 19), kucinca kwakujatana kwamubala kwakachitwa amulongo uujalilidwe (20, 21), naakuti kusanganya zintu zyobile eezyi (11).
(A) Ultraviolet/visible/near-infrared absorption spectrum, mitwe yayo ililembedwe amabala aayendelana mpawo yakabikkwa mbuli BPh peak a 775 nm. (B) Cibeela cha dichroism chizyungulukide chakabambululwa ku BChl absorbance a 805 nm. (C a D) Zibeela zya ΔA zyakasalwa kuzwa kuzipekupeku zya RC-LH114-W (C) antoomwe a RC-LH116 (D). Kuti kube kweezyanisya kubotu, mibala yoonse ilabambululwa kusika ku ∆A ya −A a 0.2 ps. (E) Mweelwe wa cytochrome c2 oxidation nikwakamana kubelesyegwa mumuni kakuli bunji bwa UQ2 (Langa mufanikisyo S8 kuti ujane data iitakwe mpindu). (F) Mumaseelo aakakomena mumumuni muniini, akati-kati naa mupati (10, 30 naa 300μMm-2 s-1, kweendelana), ma protein W alimwi a RC-L subunits mucibaka cisalazyidwe alimwi acisela ca membrane cakaandaanizyidwe. Langa mweelwe wamapulotini kwiinda mukubelesya SDS-polyacrylamide gel electrophoresis antoomwe a immunoassay (Langa mufanikisyo S9 kuti ujane data iitakwe mpindu). ziba mweelwe kweendelana a RC-LH114-W complex iisalazyidwe. Mweelwe wa RC-L ku protein-W ya complex ngu 1:1.
Ma BChls aali aawo 1 mu αβ14 loop ya RC-LH114-W (Mufanikisyo 1, A, C, a E) ali afwaafwi a RC primary donor (P) a 6.8Å kwiinda BChls zyeelene mu RC-LH116 (Mufanikisyo 1, B, D, a F, a S3); nokuba boobo, kweenda kwakufwambaana kwazibeela zyobile zitondezya kuti kuli RC-LH114-W a RC-LH116, chiindi chakutola nguzu zyakukankaminwa kuzwa ku LH1 kuya ku RC zili 40 ±4 a 44±3 ps (Mufanikisyo 2). , C a D, Cifwanikiso S4 a Table S2). Alimwi taakwe kwiimpana kupati mukutumina kwaminchina mukati ka RC (Mufanikisyo S5 amalembe aayungizyidwe). Tuladooneka kuti kweendelana kwachiindi chakutola manguzu aakati ka LH1 a RC-P nkuko akaambo kakukozyanya musinzo, mweengwe anguzu zikonzya kubelesegwa zya BChl muzibeela zyobile zya LH1. Kuboneka kuti kulanga-langa nzila yanguzu zya LH1 kusika kumusinzo musyoonto takufwambaani kwiinda kugwisya nguzu kuzwa kumasena aatali kabotu kuya ku RC. I-loop LH1 loop mu RC-LH114-W ilakonzya kunjila mukweenda kwakupya kutakwe mulimo muchiimo chakutontola loko kuti kube kulanga-langa bupange, mpawo kuli αβ14 ring conformation ndamfu aawo mpaali kupya kuzwa ku pigmentation distance ya βBChls aawo RC 1.
I RC-LH116 complex ilijisi 32 BChls antoomwe a 16 carotenoids, mpawo bubambe bwayo boonse mbubonya mbuli bwakajanwa kuzwa ku Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) DB strain (PDB07) (IDC) (297) a green algae (Blc.viridis) (PDB ID 6ET5) (10). Musule akubamba, kwakabonwa buyo kusiyana kusyoonto mumasena aa αβ heterodimers, ikapati 1-5, 15, a 16 (Chitondeezyo S6). Kubako kwa protein-W kulampindu mpati kubupange bwa LH1. Ma TMH aayo otatwe aswaangene aama loop mafwaafwi, a N-terminal kulubazu lwa lumen lwa complex mpawo C-terminal kulubazu lwa cytoplasmic (Mifanikisyo 1A a 3, A kusikila ku D). Ma protein-W taakwe maanzi manji (Chitondeezyo 3B), mpawo TMH2 antoomwe a TMH3 zilaswaangana a LH1αβ-14 kuti zipange busena buli mukati (Chitondeezyo 3, B a E kusikila ku G). Inzila yakuswaangana ilijisi Phe, Leu antoomwe a Val zyakasyeede muchibaka cha transmembrane. Eezyi zintu zyakasyaala zilibikkidwe antoomwe ama amino acid aatayandi maanzi antoomwe amabala aa αβ-14. Amwi ma polar residues alagwasya mukuswaangana, kubikkilizya a hydrogen bond aakati ka W-Thr68 a β-Trp42 atala aa complex cavity (Chitondeezyo 3, F a G). Atala lya cytoplasm, Gln34 ili afwaafwi a keto group ya αβ-14 carotenoids. Mukuyungizya, n-dodecyl β-d-maltoside (β-DDM) molecule yakakosolwa, mpawo muchila wayo uutayandi maanzi wakasika aakati ka protein-W a αβ-14, mpawo mucila wamafuta ulakonzya kuba mumubili. Twakabona kuti C-terminal resolution regions zya protein W a RCH zili afwaafwi loko, pesi tazili mukati kachiimo chakupanga zintu zimwi (Chitondeezyo 1, A a E). Nokuba boobo, inga kwaba kuswaangana mu C-terminal amino acids zitamani zya ma protein aaya obile, zikonzya kupa nzila yakujana ma protein-W muchiindi chakubunganya RC-LH114-W complex.
(A) Ma protein-W, aalangene a LH1αβ14 muchikozyano cha katuni, ajisi nketani iili mbuli musako (mubala uusalala pyu), itondezyedwe muchibeela cha electrostatic potential diagram (busena butakwe mubala wadota bujisi 0.13). (B) Mapulotini-W ayimininwa amubala uutayandi maanzi. Masena aajisi magesi atondezyedwe mubala wa cyan, masena aatayandi maanzi atondezyegwa mubala uutuba, mpawo masena aatayandi maanzi kapati atondezyegwa mubala wa orenji. (C a D) Ma protein-W atondezyedwe muchifwanikiso, mbuli mbwaabede alikozyenye a (A) (C), mpawo azungaanisigwa a 180° (D). Kweelana abusena mumulongo, zintu zisyeede zikonzya kwaandaanisigwa zibweza mubala wamubala wamubala, oomo N-terminal moili bbuluu mpawo C-terminal moili mubala uusalala pyu. (E) Mapulotini-W munzila njiyonya mbuli mu (A), antoomwe azintu zyakasyeede aawo mapulotini-W:LH1 ziimininwa amisamu iijisi zitondezyo. (F) Ma protein-W alazungaanisigwa 90° kweendelanya a (E) a LH1αβ14 muchitondezyo cha katuni, mpawo kweendelanya azintu zyakasyeede muchitondezyo cha bar. Zisyeede zyaatala kuzwa ku beta polypeptide zililembedwe. I cofactor itondezyedwe mbuli bbala lyendelana amubala wa Cifwanikiso 1, β-DDM yakabola itondezyedwe mubala wadota, mpawo oxygen itondezyedwe mubala uusalala pyu. (G) Kulanga muli (F) kwazungaanisigwa 180°, antoomwe azintu zyakasyeede zya alpha polypeptide.
Ma protein-W anjila mubusena bwa αβ heterodimer (15th muchifanikisyo 1F), aboobo alesya kujala kwa loop akusendekezya αβ heterodimers zyotatwe zyakusaanguna. Kwakabonwa kuti mweelwe mupati wa αβ-1 heterodimer yakusaanguna kweendelanya a filimu iili kabotu wakali 25° kusika ku 29° (Mufanikisyo 1, A a E), walo wakapangwa a 2° kusikila ku 8° kuseluka kwa αβ-1 mu RC A sharp contrast-LH116 (Mufanikisyo 1, B a F). I heterodimers yabili ayatatu iliimpene a 12° kusika ku 22° antoomwe a 5° kusika ku 10°, kweendelana. Akaambo kakulesyegwa kwa RC, kuseluka kwa αβ-1 takubikkilizyi αβ yabili (eeyo yeendelana a 16th αβ mu Cifwanikiso 1F), aboobo kupa kuti kube lwiindano lusalala mu LH1 ring (Cifwanikiso 1, A a E) . Akaambo kakubula ma αβ heterodimers obile, antoomwe akusweekelwa kwa BChl yone ama carotenoids obile, taakwe akati kama carotenoids aajatikizyidwe ku αβ-1 subunit, eezi zyakapa kuti kube LH114-W ring iijisi ma carotenoids ali kkumi atutatu Vegetarian antoomwe ama carotenoids aali makumi obilwe atutatwe. Kuyeeyelwa kwakusalazya kwazibeela zyobile zili mu αβ1 kusikila ku 7 zili munsi kwiinda zya LH1 loop yoonse, eezyo zinga zitondezya bupati bwa LH1 subunit iili afwaafwi a RC QB site (Chitondeezyo 4) .
Zifwanikiso zya RC-LH114-W (A a B) antoomwe a RC-LH116 (C a D) zitondezyedwe kuzwa atala/kumbali (A a B) (A a C) antoomwe abwaanda bwa Fig. 1. (B a D). Makkii aamibala atondezyedwe kululyo.
Chimwi chintu chili akati-akati chilaa mweelwe wa 1:14 ni Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Nokuba boobo, ma protein W a PufX tazikwe kukozyanya kulibonya, mpo zilaampindu mpati kuzibeela zya LH1. PufX ni TMH yomwe iijisi N-terminal cytoplasmic domain iiswaangana a cytoplasmic side ya RC-H subunit (13) aabusena bweendelana a Rps. palustris LH116αβ-16. PufX ilapanga nzila yakucincanya quinone/quinolone aakati ka RC-LH1 a cytochrome bcl complex mpawo iliwo muli Rba yoonse. sphaeroides core complex (13). Nikuba kuti nzila ya monomer-monomer ili mu Rba. I sphaeroides RC-LH1-PufX dimer ili aabusena bwakujata ma protein W mu RC-LH114-W, mpawo lwiindano lwakaletwa a PufX a protein-W luli abusena bweelene (Chitondeezyo S7A). Mulyango mu RC-LH114-W awalo ulikozyenye anzila ya quinone (8) ya Pseudomonas rosea LH1, eeyo iipangwa ama peptide aataswaangene a protein W naa PufX (Chitondeezyo S7B). Mukuyungizya, nzila ya quinone mu Blc. I emerald green LH1 yakapangwa akugwisya γ subunit yomwe (7) ili mubusena bukozyenye (Chitondeezyo S7C). Nikuba kuti zyeendelezyegwa ama protein aasiyene, kuboneka kwanzila zya quinone/quinolol mubusena bomwe mu RC-LH1 complex kulibonya mbuli chitondezyo chakusanduka, kutondezya kuti lwiindano lwakapangwa ama protein W lulakonzya kubeleka mbuli nzila ya quinone.
Mulyango uuli mu LH114-W loop uzumizya kupanga busena bwa membrane buzumanana aakati ka RC-LH114-W complex antoomwe a bulk membrane (Chitondeezyo 1G), kwiinda kuswaanganya zibeela zyobile kwiinda mumulyango wama protein mbuli muma protein. I RC-LH116 complex ilikozyene a Tch iijalidwe. Chintu chilimbuli nyeleti (22) (Chitondeezyo 1H). Mbwaanga kumwaika kwa quinone kwiinda mu membrane kulafwambaana kwiinda kumwaika kwiinda munzila nsyoonto yama protein, nzila iijulidwe ya LH114-W ilakonzya kuzumizya RC kufwambaana kwiinda nzila iijalidwe ya LH116, mpawo kumwaika kwa quinone mu RC inga kwalesyegwa. Kuti tubone naa ma protein W alakonzya kuchita kuti ma quinones asanduke kwiinda mu RC, twakachita bbazu lya cytochrome oxidation assay a ubiquinone 2 (UQ2) (chikozyano a UQ10 yachilengwaleza iijisi mucila mufwaafwi wa isoprene) (Chitondeezyo 2E). Nikuba kuti kubako kwa chelated quinone kulesya kuziba kabotu kwa Michaelis constant (RC-LH114-W antoomwe a RC-LH116 zileelela 0.2±0.1μM antoomwe a 0.5±0.2μM, kweendelana), mweelwe mupati wa RC-LH114-W ( 4.6±02-12-2) ngu s-1-1. 28±5% mpati kwiinda RC-LH116 (3.6±0.1 e-RC-1 s-1).
Twakasaanguna kuyeeyela kuti ma protein-W alajanika mu 10% ya core complex (16); aawa, mweelwe wakukkala mumumuni muniini, mumuni waakati-akati, amumumuni mupati uli 15±0.6%, 11±1% antoomwe a 0.9±0.5, kweendelana % (Chitondeezyo 2F). Kweezyanisya mweelwe wa mass spectrometry kwakatondeezya kuti kuyungizya histidine tag tiikwakacheya bunji bwa protein-W kweelanisigwa a wild-type strains (P = 0.59), aboobo eezyi zyeelelo tazili zintu zyakasandulwa zya protein-W (Chitondeezyo S10). Nokuba boobo, ooku kukkala kusyoonto kwa protein-W mu RC-LH1 complex kulakonzya kuzumizya zimwi RCs kuti zifwambaane kuvwunguluka, aboobo kuchesya kuchincha kwa quinone/quinolone mu RC-LH116 complex. Twakabona kuti mweelwe wabantu bakkala mumumuni mupati taukozyenye a data yamazubaano ya transcriptomics, eeyo itondezya kuti pufW gene expression ilatanta mumumuni muyumu (Chitondeezyo S11) (23). Kwiimpana aakati kakulemba kwa pufW akubikkwa kwa protein-W mu RC-LH1 nkunyonganya mpawo kulakonzya kutondezya kweendelezyegwa kwa protein.
Mu RC-LH114-W, 6 cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) antoomwe a 29 β-DDM molecules zyaabwa mpawo zyakabambwa muli njiyo 6 CDLs, 24 POPCs, 2 POPGs antoomwe a 21βDs. RC-LH116 (Mufanikisyo 5, A a B). Muzibeela zyobile eezyi, CDL ili afwaafwi kuba kulubazu lwa cytoplasmic lwa complex, mpawo POPC, POPG antoomwe a β-DDM zili afwaafwi kulubazu lwa luminal. Ma molecule obile aamafuta aakusanzya akagwisigwa mu αβ-1 kusikila ku αβ-6 muchibaka cha RC-LH114-W (Chitondeezyo 5A), mpawo osanwe akaandaanizyigwa muchibaka cheelene a RC-LH116 (Chitondeezyo 5B). Mafuta manji akajanwa kulubazu lumwi lwachintu, ikapati CDL, yakabungene aakati ka RC a αβ-7 kusikila ku αβ-13 (Chitondeezyo 5, A a B). Amwi mafuta aasalala alimwi ama detergents ali anze aa LH1 ring, alimwi acyl chains zipangidwe kabotu zilainda akati ka LH1 subunits, zyaambwa kuti β-DDM mu RC-LH114-W, alimwi zyaambwa kuti β-DDM mu RC A mixture of β-DDM a PO-LH16-LH16. Ziimo zikozyenye zya chelating lipids ama detergents mubupange bwesu zitondezya kuti nzintu zijatikizya mubili (Chitondeezyo S12A). Zibaka zyama molecule aakozyenye mu Tch azyalo zilijisi kweendelana kubotu. Kubomba moyo a Trv. Musyobo wa 970 RC-LH1s (Mufanikisyo S12, B kusikila ku E) (9, 12) antoomwe a hydrogen-bonding residues zya lipid head group zyakatondezya kukwabililwa kubotu mukutobelezyanya (Mufanikisyo S13), kutondezya kuti CDL iikwabilidwe iijatikizya RC (24), masena aaya alakonzya kukwabililwa mu RCH-LH1 complex.
(A a B) RC-LH114-W (A) antoomwe a RC-LH116 (B) ma peptide atondezyedwe ama katuni, mpawo mabala atondezyedwe amasamu, kubelesya mubala uuli muchifanikisyo 1. Mafuta atondezyedwe mubala uusalala pyu, mpawo ma detergents atondezyedwe mubala wa gray. UQ iijatikizyidwe ku RC QA a QB masena ili mubala wa yellow, mpawo UQ iili alikke ili mubala wa bbuluu. (C a D) Mizeezo njiyonya mbuli (A) a (B), kakunyina mafuta. (E kusikila ku G) Kulanga kupati kwa Q1(E), Q2(F) antoomwe a Q3(G) kuzwa ku RC-LH116, anketani zyakumbali zisungwaazyanya. Zilongwe zya haayidirojeni zitondezyedwe mbuli mitwe isiya.
Mu RC-LH116, zyoonse zyobile RC QA antoomwe a QB UQ, eezyo zitola lubazu mukutola ma electron munzila yakwaandaanya magesi, zilabola mumasena mozijatikizyidwe. Nokuba boobo, mu RC-LH114-W, QB quinone tiiyakamana pe mpawo iyoobandikwa munsi. Mukuyungizya ku QA antoomwe a QB quinones, ma chelated UQ molecules obile (aajanika aakati ka RC a LH1 rings) aabidwe mu RC-LH114-W kweendelana atubunga twabo tubotu (tujanika mu Q1 a Q2, kweendelana). bbazu). Cifwanikiso 5C). Zibeela zyobile zya isoprene zyapegwa kuli Q1, mpawo maapu yabunji ilagwisya 10 isoprene tails zyoonse zya Q2. Mubupange bwa RC-LH116, ma molecule otatwe aa chelated UQ10 (Q1 kusikila ku Q3, Cifwanikiso 5D) akakosolwa, mpawo ma molecule woonse alijisi bupati busalala mumusela woonse (Cifwanikiso 5, D kusikila ku G). Muzibeela zyobile, zyuuno zya mutwe wa quinone wa Q1 a Q2 zilijisi kweendelana kubotu (Chitondeezyo S12F), mpawo zilabelekela antoomwe a RC. Q1 ili kumulyango wa W gap ya RC-LH114-W (Mufanikisyo 1G a 5, C, D a E), mpawo Q2 ili afwaafwi abusena bwa QB (Mufanikisyo 5, C, D) a F). L-Trp143 antoomwe a L-Trp269 zyakasyaala zili afwaafwi kapati a Q1 a Q2 mpawo zilakonzya kupa π-stacking interactions (Chitondeezyo 5, E a F, antoomwe aChitondeezyo S12). L-Gln88, 3.0 Å kuzwa ku oxygen iili kule ya Q1, ipa chilongwe chiyumu cha haayidirojeni (Chitondeezyo 5E); eechi chisyaala chilikwabilidwe muma RC woonse kunze lyachilongwe chili kule (Chitondeezyo S13). L-Ser91 ilabikkwa mubusena bwa Thr muma RC manji (Chitondeezyo S13), ili 3.8 Angstroms kuzwa ku methyl oxygen ya Q1, mpawo ilakonzya kupa zijazyo zya haayidirojeni zitakwe nguzu (Chitondeezyo 5E). Q3 talibonyi kuti ilijisi kujatana kuli koonse, pesi ili mubusena butakwe maanzi aakati ka RC-M subunit a LH1-α subunit 5 kusikila ku 6 (Chitondeezyo 5, D a G). Q1, Q2 antoomwe a Q3 naakuti zili afwaafwi a chelated quinones azyalo zyakamanizyigwa mu Tch. Mubombe moyo, Trv. Musyobo 970 a Blc. Bupange bwa iris (9, 10, 12) butondezya busena bwakukwabilila bwa quinone mu RC-LH1 complex (Chitondeezyo S12G). Ma UQ osanwe aabola mu RC-LH116 aleendelana kabotu a 5.8±0.7 ya complex imwi aimwi yakajanwa kwiinda mukubelesya nzila ya high performance liquid chromatography (HPLC), kakuli ma UQ otatwe abola mu RC-LH114-W ali munsi a Muulo wakapimwa wa 6.2±0.3 (Fig. S14) utondeezya kuti taakwe ma Moleles aali mu UQ. chibumbwa.
Ma pseudo-symmetric L a M polypeptides aumwi ulaa ma TMH osanwe mpawo apanga heterodimer iiswaanganya BChl dimer yomwe, BChl monomers zyobile, bacteriophage (BPh) monomers zyobile, antoomwe a non- Heme iron yomwe antoomwe a UQ10 molecule yomwe naa zyobile. Kwiinda mukubako kwa zijatikizyo zya haayidirojeni akabunga ka ketone kumamanino akuyoboloka kwazyo mu Rps, ma carotenoids alabikkilizyigwa mu M-subunit, eeyo yiitwa kuti cis-3,4-dehydroorhodopin. Misyobo (25). Busena bwaanze bwa RC-H bulaangidwe ku membrane a TMH yomwe. Bupange bwa RC boonse bulikozyene azibeela zyotatwe zya RC zyamisyobo iiswaangene (mbuli Rba). sphaeroides (PDB ID: 3I4D). Ma macrocycles aa BChl a BPh, carotenoid backbone antoomwe a non-heme iron zilaswaangana mukati kakusalazya kwazibeela eezyi, mbweenya mbuli UQ10 head group ku QA site antoomwe a QB quinone ku RC-LH116 (Figure S15).
Kujanika kwazibeela zyobile zya RC zili a QB site occupancy rates yiindene kupa choolwe chipya chakulanga kuchincha kwa conformational kweendelana a QB quinone binding. Muchibaka cha RC-LH116, QB quinone ili mubusena buli afwaafwi (26), pesi kwaandaana kwa RC-LH114-W takukwe QB quinone pe. Kunyina QB quinone mu RC-LH114-W, eecho nchintu chakankamanisya nkaambo chikozyano eechi chilabeleka, kwiinda RC-LH116 chikozyano a QB quinone. Nikuba kuti zikobela zyobile zya LH1 zilapanga ma quinones aali cisambomwe, zyosanwe zilamana muchiimo cha RC-LH116 chijalilidwe, mpawo zyotatwe luzutu zilamana muchiimo cha RC-LH114-W chijalukide. Ooku kuyungizya kutamvwana kwabupange kulakonzya kutondezya kufwambaana kusanduka kwa RC-LH114-W QB sites, kufwambaana kwa quinone kinetics mu complex, akuyungizya kulangilwa kwakuzabuka LH1 loop. Tulaamba kuti kubula kwa UQ mubusena bwa RC QB bwa RC-LH114-W inga kwaba akaambo kachintu chiyumu alimwi chibeleka, mpawo busena bwa QB bwa RC-LH114-W bwakafwambaana kuzima mukucinca kwa UQ.
Kakwiina QB, kuzungaana kwa L-Phe217 kuya kubusena butakweendelani a UQ10, nkaambo kuyoopa kuti kube kudonkolana a isoprene yakusaanguna ya mucila (Chitondeezyo 6A). Mukuyungizya, kuchincha kupati kwakalibonya, ikapati helix de (helix mufwaafwi mumulongo akati ka TMH D a E) ooko L-Phe217 nkwasunkwa kuya ku QB binding pocket akuzungaana kwa L-Tyr223 (Chitondeezyo 6A) Kutyola cilongwe ca hydrogen antoomwe a M-Ap4 mpawo kujala QB busena (Chitondeezyo 6B). Helix de pivots kumatalikilo aayo, Cα ya L-Ser209 yasanduka a 0.33Å, mpawo L-Val221Cα yasanduka a 3.52Å. Kunyina kucinca kulibonya mu TMH D a E, ooko kukonzya kubikkwa antoomwe muzibeela zyoonse zyobile (Chitondeezyo 6A). Mbuli mbutuzyi, eechi ncechibaka chakusanguna mu RC chijala busena bwa QB. Kweezyanisya abupange buzulide (QB-bound) kutondeezya kuti quinone kaitanacheya, kuchincha kwabupange kuyandika kuchitila kuti injile mu quinone. L-Phe217 ilazungaana kuchita π-stacking interaction antoomwe a quinone head group, mpawo helix ilaunka anze, ikuzumizya skeleton ya L-Gly222 antoomwe a side chain ya L-Tyr223 kuti ipange hydrogen bond network iijisi hydrogen bond structure (Chitondeezyo 6, A a C).
(A) Katuni kavwumbilidwe ka hologilamu (L chain, orange/M chain, magenta) antoomwe a apo (gray) bupange, oomo zintu zipati zyakasyeede mozitondezyegwa muchikozyano cha musako. UQ10 wiimininwa amubala uusalala pyu. Muzila uujisi madoti utondezya zijatikizyo zya haayidirojeni zyakabambwa mubupange boonse. (B a C) Chitondeezyo cha apolipoprotein antoomwe abupange bwa ringi yoonse, kutondezya muya uuzwa kumbali wa L-Phe217 mubala wa lubululu a L-Tyr223 mubala uusalala pyu, kweendelana. L subunit ili orenji; zibeela zya M a H tazikwe mubala pe. (D a E) Apolipoprotein (D) antoomwe a (E) RC QB yoonse [mubala a (A) kweendelana] antoomwe a Thermophilus thermophilus PSII (mubala wa sokwe, mubala wa bbululu a plastic quinone; PDB ID: 3WU2) Align (58).
Chakutayeeyelwa, nikuba kuti zibeela zyiingi zya QB-deficient RCs zitakwe LH1 ziliwo, kucinca kwabupange kwakabonwa muchiiyo eechi tiikwakaambwa kale pe. Eezi zibikkilizya nzila yakugwisya QB kuzwa ku Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) a Rba. sphaeroides (PDB ID: 1OGV) (29), zyoonse zili afwaafwi kweendelana abupange bwazyo boonse bwa QB. Kulangisisya kabotu-kabotu kwa 3PRC kwakatondeezya kuti LDAO (Lauryl Dimethyl Amine Oxide) ma molecule aakusanzya alajatikizyigwa kumulyango wa QB, zikonzya kulesya kubamba zintu munzila iijalikide. Nikuba kuti LDAO taiboli aawo mpobede mu 1EYS naa 1OGV, aaya ma RC alibambwa kubelesya musamu omwe wakusanzya aboobo alakonzya kuchita chintu chomwe. Bupange bwamabwe aa Rba. Sphaeroides RC yakabambwa antoomwe a cytochrome c2 (PDB ID: 1L9B) ayalo ilibonya kuti ilijisi busena bwa QB bujalidwe. Nokuba boobo, muchiimo eechi, busena bwa N-terminal bwa RC-M polypeptide (buswaangana a QB binding site kwiinda mu H bond ya Tyr residue a Q helix) bulatambula bupange butali bwacilengwaleza, mpawo kuchincha kwa QB takuyungizyigwi pe (30 ). Nchintu chisyomezya nchakuti tatunabwene kusanduka kuliboobu kwa M polypeptide muchiimo cha RC-LH114-W, eecho chili afwaafwi a N-terminal region ya RC-LH116 RC. Alimwi cileelede kuzyibwa kuti nikwakamana kugwisigwa kwa LH1 antenna iibelesya musamu wakusanzya, ma apolipoprotein RCs mu PDB akamanizyigwa, alo aakagwisya ma quinone pools aamukati ama lipids aakati ka RC amukati ka LH1 ring (31, 32). RC ilazumanana kubeleka nkaambo ilabamba zyoonse zigwasya, kunze lya QB quinone iikonzya kubola, eeyo iitazungaani pe mpawo kanjikanji ilasweeka muchiindi chakulibambila (33). Mukuyungizya, zilizyibidwe kuti kugwisigwa kwa LH1 amafuta aazwa ku RC kulakonzya kuba ampindu kumilimo, mbuli kufwiinsya buumi bwa P+QB-state (31, 34, 35). Aboobo, tuyeeyela kuti kubako kwa LH1 ringi iizingulukide RC kulakonzya kubamba busena bwa QB "bwakajalwa", aboobo akukwabilila busena buli afwaafwi a QB.
Nikuba kuti apolipoprotein (kakwiina QB quinone) antoomwe abupange boonse ziiminina buyo zifwanikiso zyobile zyakusanduka kwabusena bwa QB, kutali zintu zitobelana, kuli zitondezyo zyakuti kujatana kulakonzya kuchitwa kutegwa kutabi kujatana alimwi a hydroquinone. Kuswaangana kwa quinolol a quinone afwaafwi abusena bwa QB bwa apolipoprotein kulakonzya kuba kwaandeene, ooko kupa kuti RC ikaka. Kwachiindi chilamfu kwakaambwa kuti kuchincha kwabupange kulagwasya mukujatana akucesya ma quinones. Ikukonzya kwa RCs zifwide kuchesya ma quinones nikwakamana kuzyibila mumudima kwakanyonganizyigwa (36); X-ray crystallography itondezya kuti kunyonyooka ooku kwachitika akaambo kakuti QB quinones yakaangidwe "kulamfwu" mbuli 4.5 Å kuzwa kubusena buli afwaafwi (26) , 37). Tulaamba kuti ooku kujatana kwakulamfwu ncitondezyo chaakati aakati ka apolipoprotein abupange bwa ringi yoonse, eeyo itobela kuswaangana kutaanzi a quinone akujula kwa QB site.
Musyobo wa II RC uujanika mu PSII complex yamabacteria amwi aajisi mumuni alimwi a cyanobacteria, algae alimwi azisyango ulaa bupange alimwi amulimo wakukwabilila (38). Kubamba zintu zitondezyedwe muchifanikisyo 6 (D a E) kukankaizya kukozyanya aakati ka PSII RCs a QB site ya bacterial RC complex. Ooku kweezyanisya kwachiindi chilamfu kwakali nzila yakwiiya nzila zijatikizyidwe kapati zyakujata akucheya kwa quinone. Mabbuku aakaindi akaamba kuti kucinca kwabupange kweendelana akucheya kwa PSII kwa quinones (39, 40). Aboobo, notulanga-langa kukwabililwa kwa RC, eeyi nzila yakujatikizya yatakabwene ilakonzya kubelesegwa abusena bwa QB bwa PSII RC muzisyango zijisi muya mubotu.
Rps ΔpufW (unlabeled pufW deletion) antoomwe a PufW-His (C-terminal 10x His-tagged protein-W expressed from the natural pufW locus) strains. palustris CGA009 yakapandululwa mumulimo wesu wakainda (16). Misyobo eeyi antoomwe amuzyali wa isogenic wild-type zyakajanwa mufiriji kwiinda mukubikka maseelo masyoonto aa PYE (amwi 5 g lita -1) (yakabikkidwe mu LB a -80 °C, iijisi 50% ( w/v) glycerol) ma protein, yeast extract a succinate) agar [1.5% (w/v) plate. Ipuleti yakabikkwa mumudima woonse mumudima mubusena butakwe muuya, mpawo yakabikkwa mumuni uutuba (~50 μmolm-2 s-1) yakapegwa a OSRAM 116-W halogen bulbs (RS Components, UK) kwamazuba otatwe kusikila kuli osanwe kusikila kwakazwa kabunga komwe. Kabunga komwe kakabelesegwa kusanzya 10 ml ya M22+ medium (41) yakayungizigwa a 0.1% (w/v) casamino acids (aawa yiitwa kuti M22). Chikozyano chakakomezyegwa muchiimo cha oxygen nsyoonto mumudima a 34°C akuzungaanisigwa a 180 rpm kwamahola aali makumi one atutatwe, mpawo 70 ml yachikombelo chakabikkwa mubukkale mbubonya kwamawoola aali makumi obilwe atune. Chikozyano cha semi-aerobic a volyumu ya 1 ml chibelesegwa kubikka 30 ml ya M22 mubbodela lyagilazi lya 30 ml lya universal screw-top transparent gilazi mpawo lyakabikkwa mumuni (~50μmolm-2 s-1) kwamahola aali makumi aane atutatwe anguzu zyamagineti zitakwe ntenda Stirring rod. Mpawo 30 ml yachikozyano yakabikkwa a 1 lita yachikozyano mubukkale mbubonya, eeyo yakabelesegwa kubikka malita aasika ku 9 ya chikombelo yakabikkwa mumumuni a ~200 μmolm-2 s-1 kwamahola aali 72. Maseelo aka tebulwa a 7132 RCF kwamaminiti aali 30, akabikkwa mu ~10 ml ya 20 mM tris-HCl (pH 8.0), mpawo akabikkwa a -20°C mane kusikila akayandika.
Musule akunyonyooka, bika mabwe amwi aa deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) antoomwe amapilisi obile aa Roche holoenzyme protease inhibitor (Merck, UK) kumaseelo aakabikkidwe. Mu 20,000 psi French pressure cell (Aminco, USA), maseelo akanyonganizyigwa ziindi zili 8 kusikila ku 12. Musule akugwisya maselu aatatyokede azintu zitakonzyi kunywika kwiinda mukubelesya 18,500 RCF kwamaminiti aali 15 a 4°C, chikumba chakagwisigwa kuzwa ku pigmented lysate kwiinda mukubelesya 113,000 RCF kwamahola obile a 43,000°C. Kosowa kabeela kakonzya kunywika mpawo ubike lubo chikumba chamubala mu 100 kusikila ku 200 ml ya 20 mM tris-HCl (pH 8.0) mpawo uchikozyanisye kusikila kutabi zintu ziboneka. Ichikwabilizyo chakaangidwe chakabikkwa mu 20 mM tris-HCl (pH 8.0) (Anatrace, USA) iijisi 2% (w/v) β-DDM kwawoola lyomwe mumudima a 4°C kakuli kuzungaanizyigwa. Mpawo kosanzya a 70°C kuti 150,000 RCF inyonyooke a 4°C kwawoola lyomwe kuchitila kuti ugwisye zintu zitanyonyooki.
Ichikwabilizyo cha ΔpufW chakabikkwa ku 50 ml DEAE Sepharose ion exchange column antoomwe a three column volumes (CV) ya binding buffer [20 mM tris-HCl (pH 8.0) containing 0.03% (w / v) β-DDM]. Samba kkolumini aama CV aakwabilila obile, mpawo kosanzya kkolumini amabufa obile aajisi 50 mM NaCl. I RC-LH116 complex yakagwisigwa a linear gradient ya 150 kusika ku 300 mM NaCl (muchikwabilizyo chakujatila) a 1.75 CV, mpawo chimwi chintu chakasyeede chakagwisigwa a binding buffer iijisi 300 mM NaCl a 0.5 CV. Bweza nzila yakunyonyooka aakati ka 250 a 1000 nm, kobamba kabeela kajisi mweelwe wakunyonyooka (A880/A280) wiinda ku 1 a 880 kusikila ku 280 nm, kosanzya tubili muchikwabilizyo, mpawo ubelesye nzila njiyonya alimwi a DEAE column On purification. Sanzya zibeela zili a A880/A280 ratios ziinda 1.7 antoomwe a A880/A805 ratios ziinda 3.0, kochita nkondo yatatu yakucincanya ion, mpawo ubambe zibeela zili a A880/A280 ratios ziinda 2.2 antoomwe a A880/A805 ratios ziinda 5.0.0. Ibbazu lyakasalazyigwa asyoonto lyakabikkilizyigwa kusika ku ~2 ml mu Amicon 100,000 molecular weight cut-off (MWCO) centrifugal filter (Merck, UK), mpawo lyakabikkwa a Superdex 200 16/600 size exclusion column (GE Healthcare, US) lyakajisi 200 mM NaCl buffer, mpawo lyakabikkwa mu buffer mbubonya 15. CV. Bweza ma spectra aakunyonyooka kwa bupati bwakugwisya, mpawo ubweze ma spectra aakunyonyooka a A880/A280 ratios aali 2.4 antoomwe a A880/A805 ratios aali 5.8 kusikila ku 100 A880, mpawo mpawo uzibelesye mukubamba naa kubamba cryo-TEM grid.
Ichikwabilizyo cha PufW-His chakabikkwa ku 20 ml HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) iijisi 200 mM NaCl a 0.03% (w/w)) mu IMAC buffer ( GE Healthcare). v) β-DDM]. Mulongo wakasanzyigwa ama CV osanwe aa IMAC buffer, mpawo ama CV osanwe aa IMAC buffer ajisi 10 mM histidine. Ibbazu lyamukati lyakagwisigwa kuzwa mumulongo ama IMAC buffers osanwe ajisi 100 mM histidine. Kabeela kajisi RC-LH114-W complex kalabikkilizyigwa kusika ku ~10 ml mutanki lyakavwundaulwa lijisi Amicon 100,000 MWCO filter (Merck, UK), lyakasanzyigwa ziindi zili 20 a binding buffer, mpawo lyakayungizyigwa kusika ku 25 ml Mu DEAE Sepharose column, CVs yone yakabikkwa ku b vava. Samba kkolumini aama CV one aakwabilila, mpawo ugwisye zyintu zili mu CV zili musanu atutatu a linear gradient ya 0 kusika ku 100 mM NaCl (muchikwabilizyo), mpawo ma CV one aasyeede ajisi 100 mM chikwabilizyo. A880/A805 mweelwe wiinda ku 4.6 wakabikkwa antoomwe kusika ku ~2 ml mu Amicon 100,000 MWCO centrifugal filter, mpawo wakazuzyigwa a 1.5 CV IMAC kakuchili chiindi. Bweza ma spectra aakunywa aazibeela zitabikkilizyi bupati mpawo ubikkilizye ma spectra aa A880/A280 ratios aali 2.1 antoomwe a A880/A805 ratios aali 4.6 kusikila ku 100 A880, eezyo zibelesegwa mpawo-mpawo mukubamba TEM grid naa kuyobolwa a -80°C kusikila kuyandika.
Ifuliji ya Leica EM GP yakabelesegwa kubamba ma TEM grids aatakwe kupya kapati. Ibbazu lyakasanzyigwa mu IMAC buffer kusika ku A880 ya 50, mpawo 5μl yakabikkwa a QUANTIFOIL 1.2/1.3 carbon-coated copper mesh (Agar Scientific, UK). Bikka ntalisyo a 20°C a 60% yakutontola kwamasekondi aali 30, mpawo uyifwefweme kwa masekondi otatwe, mpawo uyizimye mu ethane yamaanzi a -176°C.
Data ya RC-LH114-W complex yaka lembwa a eBIC (Electronic Bioimaging Center) (British Diamond Light Source) a Titan Krios microscope, eeyo iibeleka a 300kV, a nominal magnification ya 130,000× antoomwe anguzu zya 200kV. Gatan 968 GIF Quantum a K2 peak detector yakabelesegwa kulekkooda zifwanikiso munzila yakubala kuti ibweze data. Bupati bwa pixel buli 1.048Å, mpawo mweelwe wa dose ngu 3.83 e-Å-2s-1. Wakayobolola filimu mumasekondi ali kkumi abumwi mpawo wakayaandaanya muzibeela zili makumi aane. Belesya busena bujisi kaboni kuti ubweze ntalisyo ya microscope, mpawo ubweze mafilimu otatwe mumpako yomwe. Mukuvwula, mafilimu aali 3130 akabungikwa, akubula maanu aakati ka -1 a -3μm.
Data ya RC-LH116 complex yakabungikwa kubelesya microscope njiyonya ku Asterbury Biostructure Laboratory (University of Leeds, UK). Data yakabungikwa munzila yakubala akukomezya kwa 130 k, mpawo bupati bwa pixel bwakapimwa kusika ku 1.065 Å a dose ya 4.6 e-Å-2s-1. Filimu yaka lekkoodwa mumasekondi ali kkumi aabili mpawo yakaandaanizyigwa muzibeela zili makumi one atutatwe. Mukuvwula, mafilimu aali 3359 akabungikwa, aama defocus values ​​aakati ka -1 a -3μm.
Kubamba data koonse kuchitwa mu Relion 3.0 pipeline (42). Belesya Motioncorr 2 (43) kululamika kweenda kwamumuni kwiinda mukupima mweelwe, mpawo kobelesya CTFFIND 4.1 (44) kuzyiba CTF (contrast transfer function) parameter. Mafotomikrografu aajanika musule aakusaanguna aaya atondezyedwe muchifanikisyo 2. S16. Ichikozyano chakusala chakulichita chipangwa akusala amaanza aali 250 pixels a 1000 particles mu 250-pixel frame mpawo kakunyina kwaandaanya kwazibeela zyobile (2D), aboobo kukaka kwaandaanya ooko kweendelana akusofwaala kwasampulu naakuti kutakwe bube bulibonya. Mpawo, kusala kwakachitwa aazifwoto zyoonse, mpawo RC-LH114-W yakali 849,359 zibeela, mpawo RC-LH116 yakali 476,547 zibeela. Tubeela toonse twakasalwa twakainda muzibeela zyobile zya 2D zitakwe zyeelelo, mpawo musule akuchijaana, tubeela twakaswaangana abusena bwa kaboni, kusofwaala kwasampulu, kakunyina zitondezyo zilibonya naakuti tubeela tuvwumbilidwe loko twakakakwa, eezi zyakapa kuti kube 772,033 (90.9%) antoomwe a 359,678 (75.5) (75.5) tubeela twakabelesegwa mukusalazya 3D 3D. RC-LH114-W antoomwe a RC-LH116 kweendelana. Inzila yakusaanguna ya 3D yakapangwa kubelesya nzila ya stochastic gradient descent. Kubelesya nzila yakusaanguna mbuli chitondezyo, zibeela zyakasalwa zyaabwa muzibeela zyone mu 3D. Kubelesya nzila iili mumulongo ooyu mbuli chitondezyo, kochita kusalazya kwa 3D kuzibeela zili mumulongo mupati, mpawo ubelesye 15Å low-pass filter yakusaanguna kuvwumba busena bwa solvent, ubike 6 pixels zya soft edges, mpawo ubike ma pixels kuti ululamike Gatan K2 peak Modulation transfer function ya top detector. Ku RC-LH114-W dataset, eechi chikozyano chakusanguna chakachinchululwa akugwisya bupati bupati kumagolelo aachivwumbyo (chakagwisigwa kuzwa kubunji bwamukati mu UCSF Chimera). Izikozyanyo zyakazwa (kusala kwa RC-LH114-W antoomwe a RC-LH116 zili 3.91 antoomwe a 4.16 Å, kweendelana) zyakabelesegwa mbuli chitondezyo cha 3D classification yabili. Tubeela-beela twabelesegwa twabikkwa mukabunga ka 3D mpawo tatujisi kweendelana kupati abusena buli afwaafwi. Kuvwumbilila naa kubula zibeela ziboneka zyabupange. Nikwakainda muswaangano wabili wa 3D classification, cibeela chakajisi resolution mpati chakasalwa [Ku RC-LH114-W, cibeela comwe nzibeela zili 377,703 (44.5%), kuli RC-LH116, kuli zibeela zyobile, zyoonse antoomwe zili 260,752 (54.7% W), aawo nozibikkwa buyo zibeela zyomwe zili musule, aawo nozibikkwa buyo zyeelene. kuzyunguluka kutaanzi kakuli kusiyana kusyoonto]. Zibeela zyakasalwa zyakagwisigwa alimwi mubbokesi lya 400-pixel mpawo zyasalazyigwa a 3D. Chivwumbyo cha solvent chipangwa kubelesya 15Å low-pass filter yakusaanguna, 3 pixel map expansion antoomwe a 3 pixel soft mask. Kubelesya nzila yakusalazya CTF, kululamika kweendeenda kwacibeela comwe-comwe alimwi akusalazya CTF kwacibeela cabili, kusalazya kwa 3D, kuvwumbilila kwa solvent alimwi akubamba zintu zyakumbele zilacitwa nokwamana ntaamu imwi aimwi kutegwa kuyaambele kubambululwa bupange buzwa. Kubelesya FSC (Fourier Shell Correlation Coefficient) cut-off value of 0.143, resolutions of final models of RC-LH114-W and RC-LH116 is 2.65 and 2.80Å, respectively. I FSC curve ya model yamamanino itondezyedwe mu Cifwanikiso 2. S17.
Zibeela zyoonse zyama protein zilagwisigwa kuzwa ku UniProtKB: LH1-β (PufB; UniProt ID: Q6N9L5); LH1-α (PufA; UniProtID: Q6N9L4); RC-L (PufL; UniProt ID: O83005); RC-M (PufM; UniProt ID: A0A4Z7); RC-H (PuhA; UniProt ID: A0A4Z9); Ma protein-W (PufW; UniProt ID: Q6N1K3). SWISS-MODEL (45) yakabelesegwa kupanga nzila yakukozyanya ya RC, eeyo iijisi ma protein aatobelana aa RC-L, RC-M antoomwe a RC-H antoomwe abupange bwa Rba. sphaeroides RC yakabelesegwa mbuli chitondezyo (PDB ID: 5LSE) (46). Belesya chibelesyo cha "fit map" mu UCSF Chimera kuti ukonzye kweendelanya maapu (47), kusumpula bupange bwama protein, mpawo ubambe [4×BChl a (monomer library residue name = BCL), 2×BPh a (BPH), musyobo omwe naa yobile ya UQ10 (U10), non-heme iron yomwe (Fe) antoomwe a 34 hexadichololine (QAK)] kobelesya Coot (48) kuyungizya. Mbwaanga QAK tailiko mu laibbulali ya monomer, yakabikkwa muzibeela kubelesya eLBOW tool mu PHENIX (49).
Kuzwa waawo, LH1 yakayakwa. Kumatalikilo, chibelesyo chakuyaka chilikke mu PHENIX (49) chakabelesegwa kuyaka chibeela cha LH1 kubelesya maapu antoomwe a LH1-α antoomwe a LH1-β ma protein mbuli nzila yakunjizya. Sala LH1 subunit iizwide, uyigwisye mpawo uyibike mu Coot, ubike amaanza aako mulongo uubulide muli njiyo, mpawo usalazye zyoonse kaatana bikka BCls zyobile a (BCL) a spirilloxanthin (CRT) [kweendelana a Rps. Bunji bwa LH1 complex antoomwe a carotenoid iizyibidwe. Misyobo (17)]. Kopa LH1 subunit yoonse, mpawo ubelesye UCSF Chimera "Docking Map Tool" kuti ujatane mubusena butakwe citondezyo bwa LH1 density, mpawo uchibambulule mu Coot; koinduluka nzila kusikila zibeela zyoonse zya LH1 zyakabambwa. Kuchiimo cha RC-LH114-W, kwiinda mukugwisya bunji butabikkidwe mu Coot, ma protein alaandaanizyigwa kuzwa kuzibeela zitali zya ma protein zyakasyeede mumaapu ya USCF Chimera mpawo chibelesyo cha Autobuild chibelesegwa kubamba nzila yakusaanguna, mpawo zibeela zyakasyaala (ma protein-W) Modeling. Mu PHENIX (49). Yungizya zyoonse zitobelana zibulide kumukozyano uuzwa mu Coot (48), mpawo ubambulule amaanza woonse. Bunji butabikkidwe busyeede bulakonzya kweendelana amafuta (PDB monomer library ID of CDL = CDL, POPC = 6PL and POPG = PGT), β-DDM detergent (LMT) antoomwe a UQ10 molecules (U10). Belesya PHENIX optimization (49) antoomwe a manual optimization mu Coot (48) kuchitila kuti chikozyano chakusanguna chilondoke kusikila mweelwe wachikozyano abubotu bwachikozyano zitakonzyi kuyaambele. Kumamanino, kobelesya LocScale (50) kubamba maapu yabusena, mpawo uchite zimwi nzila zyakukozyanisya bunji bwazintu zitabikkidwe antoomwe akuchita zintu munzila iitaluleme.
Ma peptide, ma cofactors alimwi ama lipids aambi alimwi ama quinones aajatikizyidwe mukati ka densities zyawo zitondezyedwe muzifwanikiso 1 a 2. S18 kusikila ku S23. Makani aamba zyachikozyano chamamanino atondezyedwe mu Table S1.
Kusikila kwaambwa bumbi, UV/Vis/NIR yakabungikwa a Cary60 spectrophotometer (Agilent, USA) a 1 nm kuzwa ku 250 nm kusika ku 1000 nm antoomwe achiindi cha 0.1s.
Sanzya sampulu mu quartz cuvette anzila ya 2 mm kuya ku A880 ya 1, mpawo ubweze absorption spectrum aakati ka 400 a 1000 nm. Mizeezo iizungulukide yakabungikwa a Jasco 810 spectropolarimeter (Jasco, Japan) a 1 nm akati ka 400 nm a 950 nm a scan rate ya 20 nm min-1.
Mweelwe wakuzima kwa molar ulajanwa kwiinda mukusofwaazya ntalisyo yamukati kusika ku A880 iili afwaafwi kusika ku 50. Kosofwaazya 10μl volume mu 990μl binding buffer naa methanol, mpawo ubweze absorption spectrum mpawo-mpawo kuchitila kuti BChl itanyonyooki. BChl iili mukati ka musamu wa methanol yakabalilwa a 771 nm ya 54.8 mM-1 cm-1, mpawo mweelwe wakuzima wakajanwa (51). Aabanizya mweelwe wa BChl uupimidwe a 32 (RC-LH114-W) naa 36 (RC-LH116) kuti ujane mweelwe wa core complex, ooyo uubelesyegwa kuzyiba absorption spectrum ya sampulu njiyonya yakabungikwa mu buffer Extinction coefficient. kumbali. Kupima ziindi zyotatwe kwakachitwa kuli musamu umwi aumwi, mpawo mweelwe waakati-kati wa BChl Qy wakabelesegwa mukubala. Ikuzima kwa RC-LH114-W kwakapimwa a 878 nm nku 3280±140 mM-1 cm-1, mpawo kuzima kwa RC-LH116 kwakapimwa a 880 nm nku 3800±30 mM-1 cm-1.
UQ10 yakapimwa kweendelanya anzila iili mu (52). Mubufwiifwi, HPLC yakujokela musule (RP-HPLC) yakachitwa kubelesya nzila ya Agilent 1200 HPLC. Kunyonyoona aali 0.02 nmol ya RC-LH116 naa RC-LH114-W mu 50μl ya 50:50 methanol:chloroform iijisi 0.02% (w/v) ferric chloride, mpawo ubike Beckman Coulter Ultrasphere ODS 4 mml yakabambwa kale mu 1-1-1-min. 40°C mu HPLC solvent (80:20 methanol:2-propanol) aa ×25 cm column. Chita isocratic elution mu HPLC solvent kuchitila kuti ubone naa ulakonzya kunjila a 275 nm (UQ10), 450 nm (carotenoids) antoomwe a 780 nm (BChl) kwawoola lyomwe. Nkomwe ya 275 nm chromatogram kumaminiti aali 25.5 yakaswaanganizigwa, eeyo yatakajisi zimwi zintu zikonzya kubonwa. Busena busanganizidwe bubelesyegwa kubala mweelwe wa molar wa UQ10 wakagwisigwa kweendelanya a calibration curve yakabalilwa kuzwa kukubikkwa kwa zyeelelo zisalala kuzwa ku 0 kusika ku 5.8 nmol (Chitondeezyo S14). Isampulu imwi aimwi yakavwuntauzyigwa muzibeela zyotatwe, mpawo kulubizya kwakaambwa kweendelana a SD yaakati-kati.
Musamu uulaa RC-LH1 complex a Qy absorption mpati ya 0.1 wakabambilwa a 30 μM yakacesyegwa horse heart cytochrome c2 (Merck, UK) antoomwe a 0 kusika ku 50 μMUQ2 (Merck, UK). Zitondezyo zyotatwe zya 1-ml zyakabambilwa a UQ2 mpawo zyakabikkwa mubusiku boonse mumudima a 4°C kuchitila kuti zikonzye kuzyibila mumudima kakutanaba kupimwa. Inzila yakabikkwa mu OLIS RSM1000 modular spectrophotometer iijisi 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm middle antoomwe a 0.6 mm outlet slits. Chisefa chilamfu cha 600 nm chibikkidwe kumulyango wa sampulu ya phototube antoomwe a reference photomultiplier tube kuchitila kuti kutabi mumuni wakukankaminwa. Kuyoya kwakali kulanganyigwa a 550 nm achiindi chakuswaangana cha 0.15 s. Mumuni uukulwaizya ulazwa ku 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) kwiinda muntambo ya fiber optic a 90% nguzu kwiinda mu DC2200 controller (Thorlabs Ltd., UK) mpawo ulazwa kubusena bwamumuni a 90°. Mumuni wakupimya uliimpene agilazi kuti upilusye mumuni uuli woonse uutananjila muchitondezyo. langa kuyoya 10 s kakutanaba mumuni wa 50 s. Mpawo kuyoya kwakayungizyigwa kulanga kwa 60 s mumudima kuchitila kubona kusikila mpokugolela quinolol kuchesya cytochrome c23 + (Langa mufanikisyo S8 kuti ujane data iitakwe mpindu).
Data yakabelesegwa akubikka mweelwe wakusaanguna mukati ka 0.5 kusikila ku 10 s (kweendelana a UQ2) mpawo akweezyanisya mweelwe wazitondezyo zyoonse zyotatwe kuli UQ2. Bunji bwa RC-LH1 bwakabalilwa a extinction coefficient bwakabelesegwa kusandula mweelwe kuba catalytic efficiency, bwakabikkwa mu Origin Pro 2019 (OriginLab, USA), mpawo bwakabikkwa mu Michaelis-Menten model kuchitila kuzyiba mweelwe wa Km a Kcat.
Mukweela kwakunywa kwakaindi kaniini, musamu wa RC-LH1 wakasanzyigwa kusika ku ~2μM mu IMAC buffer yakajisi 50 mM sodium ascorbate (Merck, USA) antoomwe a 0.4 mM Terbutin (Merck, USA). Ascorbic acid ibelesyegwa mbuli chipaizyo cha electron donor, mpawo tert-butaclofen ibelesyegwa mbuli QB inhibitor kuchitila kuti RC donor mpati izumanane kucheya (nkokuti, tiikwe photooxidized) muchiindi choonse chakupima. Aali 3 ml ya musamu ilabikkwa ku selo iizunguluka (aasika ku 0.1 m mubwamba, 350 RPM) a 2 mm bulamfwu bwanzila yakulanga kuchitila kuti musamu uuli munzila ya laser ube achiindi chizulide chakusanduka mumudima aakati kakukankaminwa. Belesya ~100-fs laser pulses kuti uyungizye Ti: Sapphire laser system (Spectra Physics, USA) kuti ukkale chikozyano a 880 nm a 1 kHz (20 nJ ya NIR naa 100 nJ ya Vis). Kotana uyobolola data, bikka sampulu mumumuni uukkomanisya kwamaminiti aali 30. Kuyubununwa kuyoopa kuti QA itabeleki (ambweni kuchesya QA chiindi chomwe naa zyobile). Pesi kozyiba kuti eeyi nzila ilakonzya kujokolosyegwa nkaambo nikwakainda chiindi chilamfu chakuzyibila mumudima, RC ulajokela asyoonto-asyoonto kumulimu wa QA. I Helios spectrometer (Ultrafast Systems, USA) yakabelesegwa kupima transient spectra achiindi chakuchedwa cha -10 kusika ku 7000 ps. Belesya pulogilamu ya Surface Xplorer (Ultrafast Systems, USA) kuti ugwisye tubunga twa data, mpawo uswaanganya akuchita kuti kube kweendelana. Belesya pulogilamu ya CarpetView (Light Conversion Ltd., Lithuania) kubelesya data iiswaangenyi kuti ujane mibala iindene-indene iijatikizya kubola, naa kubelesya nzila iijatikizya zitondezyo zinji antoomwe abwiinguzi bwacibelesyo kutegwa zyeendelane akusanduka kwamubala omwe mu Origin (OriginLab, USA).
Mbuli mbukwaambidwe atala aawa (53), filimu ya photosynthetic iijisi LH1 complex iitakwe RC antoomwe a peripheral LH2 antenna yakabambilwa. Ichikwabilizyo chakasanzyigwa mu 20 mM tris (pH 8.0) mpawo chakabikkwa mu quartz cuvette a 2 mm optical path. I 30nJ laser pulse yakabelesegwa kukulwaizya sampulu a 540 nm achiindi chakuchedwa cha -10 kusika ku 7000 ps. Belekela data mbuli mbokwaambidwe ku Rps. mweenzinyina musamu.
Ichikwabilizyo chakakosolwa a 150,000 RCF kwamahola obile a 4°C, mpawo kuyoya kwacho a 880 nm kwakabikkwa alimwi mu 20 mM tris-HCl (pH 8.0) antoomwe a 200 mM NaCl. Kunyonyoona chikumba kwiinda mukuvwungulula asyoonto-syoonto mu 2% (w/v) β-DDM kwawoola lyomwe mumudima a 4°C. Isampulu yakasanzyigwa mu 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) kusika ku 2.5 mg ml-1 (Bio-Rad analysis). Kuyungizya kubamba kwakachitwa kuzwa munzila yakamwaigwa musule (54), kutalikila akusofwaazya 50 μg yama protein mu 50 μl ya TEAB iijisi 1% (w/v) sodium laurate (Merck, UK). Nikwakainda kusonikwa kwamasekondi aali 60, kwakacheya a 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) a 37°C kwamaminiti aali 30. Kuti uchite S-alkylation, bikka musamu a 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) mpawo uyungizye kuzwa ku 200 mM isopropanol stock solution kwamaminiti aali kkumi a temperature yamuŋanda. Kugaya kwakachitwa akuyungizya 2 μg trypsin/endoproteinase Lys-C mixture (Promega UK) mpawo kwakabikkwa a 37°C kwamahola otatwe. I laurate surfactant yakagwisigwa kwiinda mukuyungizya 50 μl ethyl acetate antoomwe a 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) mpawo akuzungaanya kwa 60 s. Kwaandaanya kwazibeela kwakasumpulwa akubelesya nzila ya centrifugation a 15,700 RCF kwamaminiti aali 5. Kweelana amalailile aabasikupanga, C18 spin column (Thermo Fisher Scientific, UK) yakabelesegwa kugwisya akugwisya munyo muchibeela chaansi chakajisi peptide. Nikwakamana kuyuma kwiinda mukubelesya nzila ya vacuum centrifugation, musamu wakanyonyoonwa mu 0.5% TFA a 3% acetonitrile, mpawo 500 ng yakavwuntauzyigwa a nanoflow RP chromatography antoomwe a mass spectrometry kubelesya nzila zyaambwa musule.
Belesya MaxQuant v.1.5.3.30 (56) kuzyiba ma protein akupima mweelwe wayo kuti uyandaule Rps. palustris proteome database (www.uniprot.org/proteomes/UP000001426). Data ya mass spectrometry proteomics yakabikkwa mu ProteomeXchange Alliance kwiinda mu PRIDE partner repository (http://proteomecentral.proteomexchange.org) munsi lya dataset identifier PXD020402.
Kuvwuntauzya a RPLC antoomwe a electrospray ionization mass spectrometry, RC-LH1 complex yakapangwa kuzwa ku Rps yamusyobo wamusyokwe. Kubelesya nzila yakamwaigwa musule (16), bunji bwama protein bwakapangwa mumaseelo aa palustris bwakali 2 mg ml-1 mu 20 mM Hepes (pH 7.8), 100 mM NaCl a 0.03% (w/v) β- (Bio-Rad analysis) ) DDM. Kweelana amulawu wabasikupanga, kobelesya 2D purification kit (GE Healthcare, USA) kugwisya 10 μg yamapulotini kwiinda mukubelesya nzila ya precipitation, mpawo unyonyoone precipitate mu 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile a 20% (v/v) maanzi. Ma microlita osanwe akavwuntauzyigwa a RPLC (Dionex RSLC) antoomwe a mass spectrometry (Maxis UHR-TOF, Bruker). Belesya MabPac 1.2×100 mm column (Thermo Fisher Scientific, UK) kwaandaanya a 60°C a 100μlmin -1, a gradient ya 85% (v / v) solvent A [0.1% (v / v) FA antoomwe a 0.02% (V/v) TFA aqueous solution] to solvent(85%)/v/v) [0.1%(v/v) FA a 0.02%(v/v) mu 90%(v/v) acetonitrile TFA] Kubelesya nzila ya electrospray ionization source anzila zyakusaanguna kwamaminiti aatandila ku 60, mass spectrometer ilajana 100 kusika ku 2750 m/z (mass-to-charge ratio). Kwiinda mukugwasyigwa a ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), bamba mass spectrum kuzibeela zya complex.
Maseelo akakomezyegwa kwamawoola aali 72 munsi lya 100 ml NF-ansi (10μMm-2 s-1), akati-kati (30μMm-2 s-1) naa atala (300μMm-2 s-1) mumuni. M22 akati-kati (M22 akati-kati oomo ammonium sulfate moitabelesyegwi mpawo sodium succinate yakanjililwa busena a sodium acetate) mubbodela lya 100 ml lya screw-top (23). Muziindi zyosanwe zya 30-s, 0.1 micron magilazi akabikkwa a volume ratio ya 1:1 kuti agwisigwe maselu mpawo akutontozyegwa aa aisi kwamaminiti aali 5. Zintu zitanyonyooki, maselu aatatyokede antoomwe ankomo zyagilazi zyakagwisigwa kwiinda mukubelesya nzila ya centrifugage a 16,000 RCF kwamaminiti aali kkumi mu benchtop microcentrifuge. Ichikwabilizyo chakaandaanizyigwa mu Ti 70.1 rotor a 100,000 RCF mu 20 mM tris-HCl (pH 8.0) a 40/15% (w/w) sucrose gradient kwamahola aali kkumi.
Mbuli mbukwakapandululwa mumulimo wesu wainda, kuvwuntauzya kwa His tag a PufW (16). Mubufwiifwi, ntalisyo iisalazyidwe (11.8 nM) naakuti chikumba chili a RC (yakajanwa kwiinda mukugusya muya uuzwa mumulengalenga mpawo kweendelanya mukuli wa gel iijisi mubala) mu 2x SDS loading buffer (Merck, UK) yakasanzyigwa ziindi zyobile. Ma protein akaandaanizyigwa a 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). Ijeli yakabikkwa mubala wa Coomassie Brilliant Blue (Bio-Rad, UK) kuti ibweze akulanga RC-L subunit. Ma protein aali a gel yabili akabikkwa mu methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) kuti achite immunoassay. I PVDF membrane yakajalilidwe mu 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 antoomwe a 5% (w / v) fulaulu wamalili, mpawo yakabikkwa antoomwe a anti-His primary antibody (mu Dilute the antibody buffer [50 mM-HCl 7.6), 150 mM NaCl a 0.05% (v/v) Tween-20] mu 1:1000 A190-114A, Bethyl Laboratories, USA) kwamawoola one. Musule akusanzya ziindi zyotatwe kwamaminiti aali musanu mubufa bwakukwabilila mubili, chikumba chakaswaanganyigwa a horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (yakasanzyigwa 1:10,000 mubuffer bwama antibody) Bikka kuti chikonzye kuzyibwa (maminiti aali 5 musule akusanzyigwa ziindi zyotatwe mubuffer bwama antibody) kubelesya WAR EST 2000. chemiluminescence substrate (Cyanagen, Italy) antoomwe a Amersham Imager 600 (GE Healthcare, UK).
Kwiinda mukuzekaula kwaabwa kwanguzu zya gel imwi aimwi iijisi mabala naakuti nzila ya immunoassay, kusanganya busena buli munsi aamutwe mpawo akubala mweelwe wanguzu zya RC-L (gel iijisi mabala) a Protein-W (immunoassay), mu ImageJ (57) Kubamba mufanikisyo. Eezyi zibeela zyakasandulwa kuba zibeela zya molar kwiinda mukuyeeyela kuti mweelwe wa RC-L ku protein-W muchikozyano cha RC-LH114-W wakali 1:1 mpawo akubambulula data yoonse kweendelana.
Kuti ujane zimwi zilembedwe muchibalo eechi, langa http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
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David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe , Mark J. Dickman, Dewey Holten, Christine Kirma, Ccoil Necoil Hunter.
Bupange bwakuboneka kabotu bwa light trap 1 complex mu reaction center bupa luzibo lupya lwa quinone dynamics.
David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe , Mark J. Dickman, Dewey Holten, Christine Kirma, Ccoil Necoil Hunter.
Bupange bwakuboneka kabotu bwa light trap 1 complex mu reaction center bupa luzibo lupya lwa quinone dynamics.
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