Ikheli lamanje: OX11 0DE, UK, i-Diamond Building, i-Harwell Science and Innovation Park, i-Dietcote, i-Oxfordshire, e-UK, i-Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
I-reaction center light-harvesting complex 1 (RC-LH1) yingcenye eqakathekileko ye-photosynthetic yebhaktheriya ephephuli. Sethule izakhiwo ezimbili ze-cryo-electron microscopy ye-RC-LH1 complex evela ku-Rhodopseudomonas palustris. Isakhiwo se-2.65-Å se-RC-LH114-W siqukethe ama-subunit we-14 we-LH1 azungeze i-RC, ephazanyiswa yiphrotheyini W, kuthi i-complex engenawo amaphrotheyini-W iphelele ukwakheka kwe-RC ebhodwe yi-RC. I-16 yeyunithi ye-LH1 evaliweko. Ukumadanisa izakhiwo lezi kunikela ukuzwisisa ngamandla we-quinone ku-RC-LH1 complex, okufaka hlangana amatjhuguluko we-conformational ebegade angakaqinisekiswa ngaphambilini lokha nakubopheka i-quinone endaweni ye-RC QB, kanye nendawo yeendawo zokubophelela i-quinone ezisizako, ezisiza ukuzidlulisela ku-RC. Isakhiwo esihlukileko sephrotheyini ye-W sivimbela ukuvalwa kwe-LH1 loop, ngaleyindlela kwakha umzila wokurhabisa ukutjhugululwa kwe-quinone/quinolone.
Amandla anikelwa yi-photosynthesis angakghona ukugcina yoke ipilo ephasini, begodu anamandla amakhulu wethekhnoloji yelanga. Lokha nakukhuthazwa ukwenziwa kweenthombe ephasini loke, amabhaktheriya weenthombe ezibubende nawo atjengisa iindlela ezihlukahlukeneko zamandla kanye namakghono wokugaya ukudla. Zingabalekela i-photosynthesis begodu zikhule njenge-heterotrophic amagciwane ebumnyameni, zingalungisa i-nitrogen ne-carbon dioxide, zikhiqize i-hydrogen, begodu zibhidlize amakhompawundi we-aromatic (1-3). Ukuze kunikelwe amandla eenkambisweni lezi, ukukhanya kufanele kutjhugululwe msinya begodu ngendlela ephumelelako kube mamandla wamakhemikhali. Ikambiso le ithoma lokha i-antenna yokubamba ukukhanya nayidonsa ukukhanya bese idlulisela amandla avalelekileko esikhungweni sokuphendula (RC), ngaleyo ndlela kuthoma ukuhlukaniswa kwetjhaji (4 – 7). Iyunithi eyisisekelo ye-photosynthesis emagciwaneni we-phototrophic abubende yakhiwe mhlobo we-2 RC, ebhodwe yi-light-harvesting complex 1 (LH1), eyakha i-RC-LH1 core complex. I-LH1 yakhiwa ziinqhema zama-αβ heterodimers, enye nenye yazo ibophelela ama-molecule amabili we-bacteria chlorophyll (BChl) a kanye ne-carotenoid eyodwa nofana ezimbili (8-12). I-antenna ye-LH1 elula khulu inama-αβ heterodimers ali-16 nofana ali-17 abhode i-RC (9-13) nge-loop evaliweko, kodwana kezinye iinhlanganisela eziqakathekileko, ama-transmembrane peptides aphazamisa ukuragela phambili kwe-LH1 ezungezeko, ngaleyo ndlela akhuthaza ukusabalala kwe-quinol/quinone hlangana ne-RC ne-cytochrome bc complex (1111, 13). Isitjalo esibubende esiphilako esiphilako i-Rhodopseudomonas (Rps.) sisilwana esiyimodeli esikwazi ukuzwisisa ukudluliswa kwamandla nama-elekthronikhi okusekela i-photosynthesis. Isakhiwo sokuthoma sekristalu se-Rps. Imodeli ye-palustris RC-LH1 complex yi-RC, ebhodwe zii-15 heterodimeric LH1 loops, eziphazanyiswa yiphrotheyini engaziwako ebizwa nge-“Protein W” (14). Iphrotheyini-W ngemva kwalokho yahlonzwa njenge-RPA4402, okuyiphrotheyini engakaqalwa ye-10.5kDa enama-transmembrane helices amathathu abikezelweko (TMH) (16). Siphakamisa ukubiza kabutjha iphrotheyini ye-rpa4402 ekhoda iphrotheyini W ibe yi-pufW ukwenzela bona ivumelane nendlela yokubiza esetjenziselwa amajini akhoda ama-subunits we-RC-L, M (pufL, pufM) kanye ne-LH1α, β (pufA, pufB). Ngokukarisako, iphrotheyini-W ikhona kwaphela ku-10% ye-RC-LH1, okuveza i-Rps. i-palustris ikhiqiza iinhlanganisela ezimbili ezihlukileko ze-RC-LH1. Lapha, sibika izakhiwo eziphezulu ze-cryo-EM (cryo-EM) zama-core complex amabili, enye enephrotheyini W kanye ne-14 αβ heterodimers, enye ngaphandle kwephrotheyini W kanye ne-16 Heterodimer LH1 loop evaliweko. Isakhiwo sethu simelela ukutjhuguluka kwesinyathelo ekuzwisiseni i-RC-LH1 eyinkimbinkimbi ye-Rps. palustris, ngombana sihlaziye inani elifanako le-variant ngayinye begodu sinesinqumo esaneleko sokwabela ngokucacileko i-peptide ngayinye kanye nama-pigment abophekileko kanye nama-lipid ahlobene nama-quinones. Ukumadanisa izakhiwo lezi kutjengisa bona amaphrotheyini amathathu we-TMH-W angazange atholakale kiyo nanyana ngiyiphi enye i-RC-LH1 complex bekube nje akhiqiza isiteshi se-quinone ukurhabisa ukutjhugululwa kwe-quinone/quinolone. Inani leendawo ezilondoloziweko zokubophelela i-lipid ne-quinone zikhonjiwe, begodu siveze ukutjhuguluka okutjha ngemva kokuhlanganiswa kwe-quinone ne-RC, okungaba kuhle ku-photosystem II (PSII) RC yezinto eziphilako ezine-oksijini. Iindaba esizitholileko zinikela ngokuzwisisa okutjha nge-kinetics yokubopha kwe-quinone/quinolone nokutjhinisana ku-RC-LH1 core complex ye-purple phototrophic bacteria.
Ukuze kube lula ukufunda okunabileko kweenhlanganisela ezimbili ezitholakala ku-Rps. palustris, sihlukanisa i-RC-LH1 ngayinye ngeendlela ze-biochemical. Iphrotheyini ye-W-deficient complex (lapha ebizwa ngokuthi yi-ΔpufW) yahlanjululwa emhlobeni ongenayo isakhi sofuzo se-pufW (16), begodu yi-RC-LH1 yinye kwaphela engakhiqizwa. Iphrotheyini equkethe i-W yenziwa mhlobo. Iphrotheyini W yomhlobo lo itjhugululwa ngethegi ye-10x His ekupheleni kwayo kwe-C, ukwenzela bona iphrotheyini equkethe iphrotheyini W ingahlanganiswa kuhle nephrotheyini W engekho khulu ngokungakhambi insimbi. I-complex ihlukaniswe kuhle (16) I-Affinity Chromatography (IMAC).
Njengombana kutjengisiwe emfanekisweni 1, zombili iinhlanganisela ziqukethe amayunithi amathathu we-RC (RC-L, RC-M kanye ne-RC-H) abhodwe yi-antenna ye-LH1. Isakhiwo se-2.80-A se-complex engenaprotheyini-W sitjengisa ama-αβ heterodimers ayi-16, okwenza i-LH1 loop evaliweko ezungeze i-RC, lapha ebizwa ngokuthi yi-RC-LH116 complex. Isakhiwo esiyi-2.65Å se-protein-W-equkethe i-complex sine-14-heterodimer LH1 ephazanyiswe yi-protein-W, lapha ebizwa ngokuthi yi-RC-LH114-W.
(A no-B) Ukujanyelwa kwephasi kwekhompawundi. (C no-D) Imibala ehlanganisiweko evezwe ngeentonga. (E no-F) Iinhlanganisela ezibonwe ukusuka ebusweni be-cytoplasmic zinama-peptides nama-subunits we-LH1 avezwe kumakhathuni, begodu zinomboro ngokuya ngewashi ukusuka esikhaleni seprotheyini-W [ngokuhambisana nokunombolo kwe-Rba. sphaeroides complex (13)]. Ku-LH1-α, umbala wephrotheyini encani mthubi; nge-LH1-β, umbala wephrotheyini encani uluhlaza okwesibhakabhaka; ngephrotheyini-W, iphrotheyini ibomvu; nge-RC-H, yi-cyan; nge-RC-L, yi-Orentji; ye-RC-M, umbala omnyama. Ama-cofactors amelelwa ziintonga, okuluhlaza kujamele ama-molecule we-BChl kanye ne-BPh, umbala ophephuli ujamele ama-carotenoids, begodu ophuzi ujamele ama-molecule we-UQ10. (G no-H) Umbono okhulisiweko wegebe lamaphrotheyini-W esifundeni esilinganako se-RC-LH114-W (G) kanye ne-RC-LH116 (H). Ama-cofactors atjengiswa ngendlela yokuzalisa isikhala, i-quinone e-chelated itjengiswa ngombala oluhlaza okwesibhakabhaka. Isikhala sephrotheyini-W sivezwe ngomugqa ohlaza okwesibhakabhaka ku-(G), begodu imigodi emincani lapho i-quinone/quinolol irhatjheka khona ku-LH116 ring ivezwe ngomuda omnyama ku-(H).
Umfanekiso 1 (A no-B) utjengisa i-RC ebhodwe ziinqophiso ezivulekileko nofana ezivaliweko ze-LH1αβ heterodimers, ngayinye yazo ibopha ama-BChl amabili ne-carotenoid yinye (Umfanekiso 1, C no-D). Iimfundo zangaphambilini zitjengise bona i-Rps yi-LH1 eyinkimbinkimbi. Endleleni ye-biosynthetic ye-spirulina xanthin, imihlobo le inemihlobo ehlangeneko yama-carotenoids (17). Kodwana, i-spiropyrroxanthin yi-carotenoid ephambili begodu ukuminyana kwayo kuyanelisa. Ngalokho-ke, sakhetha ukumodela i-spiroxanthin kizo zoke iindawo zokubophelela i-LH1. I-alpha ne-beta polypeptides zii-TMH ezilodwa ezineendawo ezifitjhani zangaphandle (Umfanekiso 1, A, B, E, no-F). Nanyana ukuminyana kweensalela ezili-17 e-C-terminus kungabonakali, i-alpha polypeptide yahlukaniswa ukusuka ku-Met1 ukuya ku-Ala46 kizo zombili iinhlanganisela. I-β polypeptide yehliswa ukusuka ku-Gly4 ukuya ku-Tyr52 ku-RC-LH116, begodu ukusuka ku-Ser5 ukuya ku-Tyr52 ku-RC-LH114-W. Akukho ukuminyana kwama-3 nofana ama-4 we-N-terminal nofana ama-13 we-C-terminal residues okwabonwa (Umfanekiso S1). Ukuhlaziywa kwe-mass spectrometry ye-RC-LH1 ehlangeneko elungiselelwe emhlobeni we-wild-type kutjengisa bona indawo engekho bekungumphumela wokuhlukaniswa okuhlukileko kwama-peptide la (Umfanekiso S1 no-S2). I-N-terminal formylation ye-α-Met1 nayo yabonwa (f). Ukuhlaziywa kutjengise bona i-α-peptide inama-residues fMet1 ukuya ku-Asp42/Ala46/Ala47/Ala50, begodu i-β-peptide inama-residues we-Ser2 ukuya ku-Ala53, okuvumelana kuhle nebalazwe lokuminyana kwe-EM lokutjhisa okuphasi.
Ukuhlanganiswa kwe-α-His29 ne-β-His36 kwenza bona i-BChls iqalene; i-αβ heterodimer ngayinye ihlangana nabomakhelwana bayo ukwakha iluphu evulekileko (RC-LH114-W) nofana iluphu evaliweko (RC-LH116) ezungeze i-RC I-exciton coupled pigment array (Umfanekiso 1, C no-D). Nayiqathaniswa ne-877 nm band ye-RC-LH114-W, i-880 nm yokumuncwa okubomvu kwe-RC-LH116 yi-3 nm (Umfanekiso 2A). Kodwana, i-circular dichroism spectrum ifana (Umfanekiso 2B), okutjengisa bona nanyana kunomehluko ocacileko hlangana nama-loop avulekileko navaliweko, indawo yendawo ye-BChls iyafana khulu. Ukutjhuguluka okubomvu kokumuncwa kungaba mphumela wokunciphisa ukunyakaza okutjhisako nokukhuphuka kokuzinza ku-loop evaliweko (18, 19), ukutjhuguluka kokuhlanganiswa kwe-pigment okubangelwa yi-loop evaliweko (20, 21), nofana ukuhlanganiswa kwemiphumela emibili (11).
(A) I-ultraviolet/ebonakalako/eseduze ne-infrared, iinqongo zayo zitshwaywe ngemibala ehambelana nazo begodu zenziwe zibe sezingeni eliphezulu le-BPh ku-775 nm. (B) I-circular dichroism spectrum ejwayelekileko ukuya ku-BChl absorbance ku-805 nm. (C no-D) I-ΔA spectra ekhethiweko evela ku-time-resolved absorption spectra ye-RC-LH114-W complex (C) kanye ne-RC-LH116 complex (D). Ukumadanisa ngcono, woke ama-spectra ajwayelekile ku-∆A ye-−A ku-0.2 ps. (E) Izinga lokutjhuguluka kwe-cytochrome c2 ngemva kokukhanya lapho kukhona amazinga ahlukahlukeneko we-UQ2 (qala isithombe S8 ukuthola idatha engakalungiswa). (F) Emaseli akhuliswe ngaphasi kokukhanya okuphasi, okuphakathi nofana okuphezulu (10, 30 nofana 300μMm-2 s-1, ngokulandelana), iphrotheyini W kanye nama-subunits we-RC-L ku-complex ehlanjululweko kanye nesilinganiso se-membrane esihlukanisiweko. Hlola izinga lamaphrotheyini nge-SDS-polyacrylamide gel electrophoresis kanye ne-immunoassay (qala isithombe S9 ukuthola idatha engakalungiswa). Hlola isilinganiso esihlobene ne-RC-LH114-W ehlanjululweko. I-stoichiometric ratio ye-RC-L ukuya kuphrotheyini-W ye-complex yi-1:1.
Ama-BChls asesikhundleni 1 ku-αβ14 loop ye-RC-LH114-W (Umfanekiso 1, A, C, no-E) aseduze nomnikeli we-RC (P) nge-6.8Å ukudlula ama-BChls alinganako ku-RC-LH116 (Umfanekiso 1, B, D, no-F, no-S3); nanyana kunjalo, ukumuncwa kwesikhatjhana kwama-complex amabili kutjengisa bona ku-RC-LH114-W kanye ne-RC-LH116, isikhathi sokudlulisa amandla wokuvusa ukusuka ku-LH1 ukuya ku-RC siyi-40 ± 4 kanye ne-44 ± 3 ps (Umfanekiso 2). , C no-D, Umfanekiso S4 kanye neThebula S2). Akukho mehluko oqakathekileko ekudluliseleni kwe-elekthronikhi ngaphakathi kwe-RC (Umfanekiso S5 kanye nomtlolo wokungezelela ohlobene nawo). Sisola bona ukuhlobana okuseduze kwesikhathi sokudlulisa amandla hlangana ne-LH1 ne-RC-P kungenxa yebanga elifanako, i-engele kanye namandla angaba khona we-BChl enengi ema-loop amabili we-LH1. Kubonakala ngasuthi ukuhlola iphetheni yamandla we-LH1 ukufikelela ibanga eliphasi akukhawulezi ukudlula ukudluliswa kwamandla okuqondileko ukusuka eendaweni ezingakalungi ukuya ku-RC. I-loop ye-LH1 evulekileko ku-RC-LH114-W nayo ingathola ukunyakaza okungakaqakatheki ngaphasi kweemeko eziphasi zokuhlaziywa kwesakhiwo, begodu kune-αβ14 ring conformation ede emazingeni womtjhiso wegumbi ukusuka ebangeni lokubala le-βBChls endaweni ye-RC 1.
I-RC-LH116 complex inama-BChls ama-32 nama-carotenoids ali-16, begodu ukuhleleka kwayo koke kuyafana nalokho okutholwe ku-Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) DB strain (PDB07) (PDB) (297)) kanye ne-algae eluhlaza (Blc.viridis) (PDB ID 6ET5) (10). Ngemva kokuqondisa, kwabonwa ukuphambuka okuncani kwaphela eendaweni zama-αβ heterodimers, khulukhulu 1-5, 15, no-16 (Umfanekiso S6). Ukuba khona kwephrotheyini-W kunomthelela omkhulu esakhiweni se-LH1. Ama-TMH wayo amathathu ahlanganiswe ngama-loop amafitjhani, nge-N-terminal ehlangothini le-lumen ye-complex kanye ne-C-terminal ehlangothini le-cytoplasmic (Imifanekiso 1A no-3, A ukuya ku-D). Iphrotheyini-W ayithandi amanzi (Umfanekiso 3B), begodu i-TMH2 ne-TMH3 zisebenzisana ne-LH1αβ-14 ukwakha ubuso be-transmembrane (Umfanekiso 3, B no-E ukuya ku-G). I-interface yakhiwe khulu ziinsalela ze-Phe, Leu kanye ne-Val esifundeni se-transmembrane. Iinsalela lezi zifakwe ngama-amino acid angafuni amanzi kanye nama-αβ-14 amabala. Ezinye iinsalela ze-polar nazo zifaka isandla ekusebenzisaneni, kufaka hlangana ukuhlobana kwe-hydrogen hlangana ne-W-Thr68 ne-β-Trp42 ebusweni bomgodi ohlangahlangeneko (Umfanekiso 3, F no-G). Phezu kwe-cytoplasm, i-Gln34 iseduze nesiqhema se-keto se-αβ-14 carotenoids. Ukungezelela, i-n-dodecyl β-d-maltoside (β-DDM) molecule yararululwa, begodu umsila wayo ongathandi amanzi wanweba waya lapho kuhlangana khona iphrotheyini-W ne-αβ-14, begodu umsila we-lipid ungatholakala emzimbeni. Siphinde satjheja bona iindawo ze-C-terminal zephrotheyini W ne-RCH ziseduze khulu, kodwana azikho ngaphakathi kwesilinganiso sokwakha ukusebenzisana okuthileko (Umfanekiso 1, A no-E). Kodwana, kungaba nokusebenzisana kwama-amino acid we-C-terminal angakalungiswa wamaphrotheyini amabili la, okunganikela indlela yokuqatjhwa kwamaprotheyini-W ngesikhathi sokuhlanganiswa kwe-RC-LH114-W.
(A) Iphrotheyini-W, eqalene nesisetjenziswa se-LH1αβ14 ngendlela yekhathuni, ineketanga eliseceleni elibunjwe njengenduku (elibomvu), elitjengiswa engcenyeni yomdwebo we-electrostatic potential (indawo empunga ebonakalako enezinga le-contour le-0.13). (B) Iphrotheyini-W emelelwa yindawo enombala ongathandi amanzi. Iindawo ezine-polar nezine-charge zitjengiswa ngombala we-cyan, iindawo ezingafuni amanzi zitjengiswa ngombala omhlophe, begodu iindawo ezithanda amanzi khulu zitjengiswa ngombala we-orenji. (C no-D) Iphrotheyini-W evezwe kukhathuni, ukuqondiswa kwayo kuyafana neku-(A) (C), begodu ijikelezwe nge-180° (D). Ngokuya ngobujamo bokulandelana, iinsalela ezihlukileko zithatha umbala womnyama, lapho i-N-terminal ihlaza satjani bese i-C-terminal ibomvu. (E) Iphrotheyini-W ngombono ofanako naku-(A), begodu iinsalela ezise-interface yephrotheyini-W:LH1 zijanyelwe ziintonga ezinamatshwayo anamatheleko. (F) Iphrotheyini-W ijikeleziswa ngo-90° ngokuya ngo-(E) kanye ne-LH1αβ14 ekujanyelweni kwekhathuni, begodu ngokuya ngokuya kweensalela ze-interface ekujanyelweni kwebha. Iinsalela ezilengako ezivela ku-beta polypeptide zifakwe amalebula. I-cofactor itjengiswa njengebha ehambelana nombala we-Figure 1, i-β-DDM ebolileko itjengiswa ngombala ompunga, begodu i-oksijini itjengiswa ngombala obomvu. (G) Umbono ku-(F) ujikelezwe nge-180°, ngeensalela ezibonakalako ze-alpha polypeptide elebuliweko.
Iphrotheyini-W ithatha indawo ye-αβ heterodimer (ye-15 kuMfanekiso 1F), ngaleyo ndlela ivimbela ukuvalwa kwe-loop nokutjhinga ama-αβ heterodimer amathathu wokuthoma. Kwatjhejwa bona i-engeli ephezulu yokuthambekela kwe-αβ-1 heterodimer yokuthoma ngokuya ngefilimu ejayelekileko bekuyi-25° ukuya ku-29° (Umfanekiso 1, A no-E), eyakhiwa kutjhinga okungu-2° ukuya ku-8° kwe-αβ-1 ku-RC A sharp contrast-LH116 (Umfanekiso 1, B no-F). I-heterodimers yesibili neyesithathu ithambekele ku-12° ukuya ku-22° kanye no-5° ukuya ku-10°, ngokulandelana. Ngebanga lokuvimbela kwe-RC, ukutjhinga kwe-αβ-1 akufaki i-αβ yesibili (ehambelana ne-16th αβ kuMfanekiso 1F), ngaleyo ndlela kwakha isikhala esicacileko ku-LH1 ring (Umfanekiso 1, A no-E) . Ngebanga lokutlhogeka kwama-αβ heterodimers amabili, okukhambisana nokulahlekelwa kwama-BChl amane nama-carotenoids amabili, akukho nanye yama-carotenoids abophelela ku-αβ-1 subunit etjhugulukileko, okurholela ekutheni kube neringi ye-LH114-W enama-carotenoids ali-13 we-Vegetarian kanye nama-28 BChls. Iinlinganiso zendawo zama-complex amabili ezindaweni ze-αβ1 ukuya ku-7 ziphasi kunalezo ze-LH1 loop, okungatjengisa ubujamo be-LH1 eduze nendawo ye-RC QB (Umfanekiso 4) .
Iinthombe ze-RC-LH114-W (A no-B) kanye ne-RC-LH116 (C no-D) zitjengiswa ukusuka ekubukeni okufanako phezulu/eceleni (A no-B) (A no-C) kanye nendawo yomgodi we-Fig. 1. (B no-D). Iinkinobho ezinemibala zitjengiswe ngesidleni.
Enye into ene-core complex ene-stoichiometric ratio ye-1:14 yi-Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Kodwana, iphrotheyini W ne-PufX azinakho ukufana okusobala, begodu zinomthelela omkhulu ezakhiweni zazo ze-LH1. I-PufX yi-TMH eyodwa ene-N-terminal cytoplasmic domain esebenzisana ne-cytoplasmic side ye-RC-H subunit (13) endaweni ehambelana ne-Rps. palustris LH116αβ-16. I-PufX yenza umzila wokuthengiselana kwe-quinone/quinolone hlangana ne-RC-LH1 kanye ne-cytochrome bcl complex begodu ikhona kiyo yoke i-Rba. sphaeroides core complex (13). Nanyana isikhombisi se-monomer-monomer sise-Rba. I-sphaeroides RC-LH1-PufX dimer itholakala endaweni yokubophelela iphrotheyini W ku-RC-LH114-W, begodu isikhala esibangelwa yi-PufX nephrotheyini-W sisendaweni efanako (Umfanekiso S7A). Isikhala ku-RC-LH114-W siphinde sihambisane ne-hypothetical quinone channel (8) ye-Pseudomonas rosea LH1, eyakhiwe ngama-peptide angahlobene nephrotheyini W nofana i-PufX (Umfanekiso S7B). Ngaphezu kwalokho, isiteshi se-quinone ku-Blc. I-LH1 eluhlaza satjani eyakhiwe ngokungafaki iyunithi eyodwa ye-γ (7) itholakala endaweni efanako (Umfanekiso S7C). Nanyana kulawulwa maprotheyini ahlukileko, ukuvela kwamatjhaneli we-quinone/quinolol endaweni efanako ku-RC-LH1 kubonakala kusibonelo sokuziphendukela kwemvelo okuhlangeneko, okutjengisa bona isikhala esenziwe yiphrotheyini W singasebenza njengetjhaneli ye-quinone.
Isikhala se-LH114-W loop sivumela ukwakheka kwesifunda se-membrane esiragela phambili hlangana nesikhala sangaphakathi se-RC-LH114-W complex kanye ne-bulk membrane (Umfanekiso 1G), kunokobana kuhlanganiswe ama-domain amabili nge-pore yamaphrotheyini njengamaphrotheyini. I-RC-LH116 ifana ne-Tch evaliweko. Inkimbinkimbi efana nenaliti (22) (Umfanekiso 1H). Njengombana ukusabalala kwe-quinone nge-membrane kukhawuleza ukudlula ukusabalala ngephrotheyini encani, iluphu evulekileko ye-LH114-W ingavumela ukujika okurhabako kwe-RC ukudlula iluphu evaliweko ye-LH116, begodu ukusabalala kwe-quinone ngaphakathi kwe-RC kungaba mkhawulo khulu. Ukuhlola bona iphrotheyini W ithinta na ukutjhugululwa kwama-quinones nge-RC, senza i-cytochrome oxidation assay ku-ubiquinone 2 (UQ2) (i-analogue ye-UQ10 yemvelo enomsila omfitjhani we-isoprene) (Umfanekiso 2E). Nanyana ubukhona be-quinone e-chelated kuvimbela ukunqunywa okunembileko kwe-Michaelis constant (RC-LH114-W kanye ne-RC-LH116 zilungele i-0.2±0.1μM kanye ne-0.5±0.2μM, ngokulandelana), izinga eliphezulu le-RC-LH114-W ( 4.6±0.21-2-1) li-e-s-. 28±5% ekulu kune-RC-LH116 (3.6±0.1 e-RC-1 s-1).
Ekuthomeni besilinganise bona iphrotheyini-W ikhona ku-10% ye-core complex (16); lapha, amazinga wokuhlala wamaseli wokukhula wokukhanya okuncani, okukhanyako okuphakathi, nokukhanya okukhulu ngu-15±0.6%, 11±1% kanye no-0.9±0.5, ngokulandelana % (Umfanekiso 2F). Ukumadanisa okulinganiselweko kwe-mass spectrometry kutjengise bona ukungezelelwa kwethegi ye-histidine akuzange kwehlise inani elihlobeneko leprotheyini-W nayiqathaniswa nemihlobo ye-wild-type (P = 0.59), ngalokho amazinga la akusiyo into eyakhiweko yephrotheyini-W etjhugululiweko (Umfanekiso S10). Kodwana, ukuhlala okuphasi kwamaphrotheyini-W ku-RC-LH1 kungavumela amanye ama-RC bona aphenduke ngezinga elirhabako, ngaleyo ndlela kunciphise ukutjhugululwa okubuthakathaka kwe-quinone/quinolone ku-RC-LH116. Sinanzelele bona izinga eliphezulu lokuhlala emkhanyweni alihambisani nedatha yakamuva ye-transcriptomics, etjengisa bona ukuvezwa kwezakhi zofuzo ze-pufW kukhuphuka ngaphasi kokukhanya okunamandla (Umfanekiso S11) (23). Umehluko phakathi kokutlolwa kwe-pufW nokufakwa kwephrotheyini-W ku-RC-LH1 kuyadida begodu kungatjengisa ukulawulwa okuyinkimbinkimbi kwephrotheyini.
Ku-RC-LH114-W, i-6 cardiolipin (CDL), i-7 phosphatidylcholine (POPC), i-1 phosphatidylglycerol (POPG) kanye nama-29 we-β-DDM amamolekhuli abelwe begodu amodelwe kiwo ama-CDL ama-6, ama-POPC ama-24, ama-POPG ama-2 kanye nama-21 βD. RC-LH116 (Umfanekiso 5, A no-B). Ezakhiweni ezimbili lezi, i-CDL ipheze ibekwe ngehlangothini le-cytoplasmic ye-complex, kuthi i-POPC, i-POPG kanye ne-β-DDM ibekwe khulu ngehlangothini le-luminal. Amamolekhyuli amabili we-lipid ne-detergent ahlukaniswe endaweni ye-αβ-1 ukuya ku-αβ-6 ye-RC-LH114-W (Umfanekiso 5A), begodu amahlanu ahlukaniswe endaweni efanako ye-RC-LH116 (Umfanekiso 5B ). Amafutha amanengi atholakala ngakelinye ihlangothi le-complex, khulukhulu i-CDL, ebuthelelwe hlangana ne-RC ne-αβ-7 ukuya ku-αβ-13 (Umfanekiso 5, A no-B). Amanye amafutha nama-detergent alungiswe ngokwesakhiwo atholakala ngaphandle kwendandatho ye-LH1, begodu amaketango we-acyl alungiswe kuhle anweba hlangana nama-subunits we-LH1, akhethwe njenge-β-DDM ku-RC-LH114-W, begodu ahlathululwe njenge-β-DDM ku-RC A umxube we-β-DDM ne-PO-LH116. Iindawo ezifanako zamafutha we-chelating nama-detergent esakhiweni sethu zitjengisa bona ziindawo zokubophelela ezihlobene nomzimba (Umfanekiso S12A). Iindawo zamamolekhyuli alinganako ku-Tch nazo zinokuvumelana okuhle. Ubumnene noTrv. I-Strain 970 RC-LH1s (Umfanekiso S12, B ukuya ku-E) (9, 12) kanye nama-hydrogen-bonding residues wesiqhema se-lipid head atjengise ukulondolozwa okuhle ekulandeleleni (Umfanekiso S13), okutjengisa bona i-CDL elondoloziweko ebophelela ku-RC (24), amasayithi la angagcinwa ku-RCH-LH1.
(A no-B) Ama-peptide we-RC-LH114-W (A) kanye ne-RC-LH116 (B) ajanyelwe ngamakhathuni, begodu ama-pigment amelelwa ziintonga, kusetjenziswa umbala kuMfanekiso 1. Amafutha atjengiswa ngombala obomvu, begodu iinsetjenziswa zokuhlanza zitjengiswa ngombala ompunga. I-UQ ebotjhelelwe kumasayithi we-RC QA ne-QB mthubi, kuthi i-UQ ehlukileko iluhlaza okwesibhakabhaka. (C no-D) Imibono efanako no-(A) no-(B), ngaphandle kwamafutha. (E ukuya ku-G) Umbono okhulisiweko we-Q1(E), Q2(F) kanye ne-Q3(G) ukusuka ku-RC-LH116, ngamaketanga aseceleni athonya omunye nomunye. Amabhondi we-hydrogen atjengiswa njengemigqa emnyama.
Ku-RC-LH116, zombili i-RC QA ne-QB UQ, ezihlanganyela ekudlulisweni kwe-elekthronikhi emsebenzini wokuhlukaniswa kwetjhaji, ziyabola eendaweni zazo zokubopha. Kodwana, ku-RC-LH114-W, i-QB quinone ayikalungiswa begodu izokucocwa ngayo ngokunabileko ngenzasi. Ngaphezu kwama-QA kanye ne-QB quinones, ama-molecule amabili we-UQ (atholakala hlangana namasongo we-RC ne-LH1) abelwe esakhiweni se-RC-LH114-W ngokuya ngamaqembu wawo wehloko alungiswe kuhle (atholakala ku-Q1 ne-Q2, ngokulandelana). isikhala). Umfanekiso 5C). Amayunithi amabili we-isoprene abelwe ku-Q1, begodu imephu yokuminyana ixazulula imisila ye-isoprene eli-10 epheleleko ye-Q2. Esakhiweni se-RC-LH116, amamolekhyuli amathathu we-UQ10 (Q1 ukuya ku-Q3, Umfanekiso 5D) ararululwe, begodu woke amamolekhyuli anesisindo esicacileko kiwo woke umsila (Umfanekiso 5, D ukuya ku-G). Ezakhiweni ezimbili, iindawo zamaqembu wehloko ye-quinone ye-Q1 ne-Q2 zinokuvumelana okuhle khulu (Umfanekiso S12F), begodu zisebenzisana ne-RC kwaphela. I-Q1 itholakala emnyango we-W gap ye-RC-LH114-W (Umfanekiso 1G no-5, C, D no-E), begodu i-Q2 itholakala eduze nendawo yokubophelela i-QB (Umfanekiso 5, C, D) no-F). Iinsalela ze-L-Trp143 ne-L-Trp269 ezilondoloziweko ziseduze khulu ne-Q1 ne-Q2 begodu zinikela ngokusebenzisana okungenzeka kwe-π-stacking (Umfanekiso 5, E no-F, kanye noMfanekiso S12). I-L-Gln88, 3.0 Å ukusuka ku-oksijini ekude ye-Q1, inikela ngesibopho esinamandla se-hydrogen (Umfanekiso 5E); insalela le igcinwe kiwo woke ama-RC ngaphandle kobudlelwano obukude khulu (Umfanekiso S13). I-L-Ser91 ifakwe esikhundleni se-Thr kezinye i-RC ezinengi (Umfanekiso S13), yi-3.8 Angstroms ukusuka ku-methyl oxygen ye-Q1, begodu inganikela ngamabhondi we-hydrogen abuthakathaka (Umfanekiso 5E). I-Q3 ayibonakali inokuthintana okuthileko, kodwana isendaweni engathandi amanzi hlangana ne-RC-M subunit kanye ne-LH1-α subunit 5 ukuya ku-6 (Umfanekiso 5, D no-G). I-Q1, i-Q2 ne-Q3 nofana ama-quinone aseduze nawo sele ararululwe ku-Tch. Mnene, Trv. Umhlobo 970 kanye ne-Blc. Isakhiwo se-iris (9, 10, 12) sikhomba endaweni yokubopha ye-quinone evikelweko ku-RC-LH1 complex (Umfanekiso S12G). Ama-UQ amahlanu abolileko ku-RC-LH116 avumelana kuhle ne-5.8±0.7 ye-complex ngayinye enqunywe yi-high performance liquid chromatography (HPLC), kuthi ama-UQ amathathu abolileko ku-RC-LH114-W aphasi kunenani elilinganisiweko le-6.2±0.3 (Umfanekiso S14) litjengisa bona akunawo amamolethi we-UQ. isakhiwo.
I-pseudo-symmetric L kanye ne-M polypeptides ngayinye inama-TMH amahlanu begodu yakha i-heterodimer ehlanganisa i-BChl dimer yinye, ama-monomers amabili we-BChl, ama-monomers amabili we-bacteriophage (BPh), kanye ne-non-Heme iron kanye ne-UQ10 molecule eyodwa nofana ezimbili. Ngokuba khona kwama-hydrogen bonds ku-terminal ketone group kanye nokubuthelela kwawo okwaziwako ku-Rps, ama-carotenoids afakwa ku-M-subunit, ebizwa nge-cis-3,4-dehydroorhodopin. Imihlobo (25). Isizinda se-membrane yangaphandle ye-RC-H sibophelelwe e-membrane nge-TMH eyodwa. Isakhiwo se-RC esipheleleko sifana nama-subunit amathathu we-RC wemihlobo ehlobeneko (njenge-Rba). sphaeroides (PDB ID: 3I4D). I-macrocycles ye-BChl ne-BPh, umgogodla we-carotenoid kanye ne-non-heme iron iyahlangana ngaphakathi kwebanga lokulungiswa kwalezi zakhiwo, njengombana kwenza isiqhema sehloko se-UQ10 endaweni ye-QA kanye ne-QB quinone ku-RC-LH116 (Umfanekiso S15).
Ukutholakala kwezakhiwo ezimbili ze-RC ezinamazinga ahlukileko wokuhlala endaweni ye-QB kunikela ithuba elitjha lokuhlola amatjhuguluko angaguqukiko we-QB quinone. Ku-RC-LH116 complex, i-QB quinone itholakala endaweni ebophekileko "eseduze" (26), kodwana ukuhlukaniswa kwe-RC-LH114-W ayinayo i-QB quinone. Ayikho i-QB quinone ku-RC-LH114-W, okumangalisako ngombana i-complex iyasebenza, ukudlula i-RC-LH116 complex ene-QB quinone elungiswe ngokwesakhiwo. Nanyana amasongo amabili we-LH1 ahlanganisa ama-quinone asithandathu, amahlanu ararululwe ngokwesakhiwo ngaphakathi kwendandatho ye-RC-LH116 evaliweko, lokha amathathu kwaphela alinganiselwe ngokwesakhiwo ngaphakathi kwendandatho ye-RC-LH114-W evulekileko. Ukuphazamiseka kwesakhiwo okwandako kungatjengisa ukutjhugululwa okurhabako kwamasayithi we-RC-LH114-W QB, ukunyakaza okurhabako kwe-quinone ngaphakathi kwe-complex, kanye nokunyuka kwamathuba wokweqa i-LH1 loop. Siphakamisa bona ukutlhogeka kwe-UQ endaweni ye-RC QB ye-RC-LH114-W kungaba mphumela wendawo eyinkimbinkimbi nesebenzako, begodu indawo ye-QB ye-RC-LH114-W sele iqhwa msinyana ekubuyeleni kwe-UQ Isigaba esithileko (umnyango wendawo ye-QB uvaliwe) utjengisa ukwakheka kwalokhu kusebenza.
Ngaphandle kwe-QB, ukujikeleza okukhambisana ne-L-Phe217 ukuya endaweni engahambisani nokubopha kwe-UQ10, ngombana kuzokubangela ukutjhayisana kwendawo neyunithi yokuthoma ye-isoprene yomsila (Umfanekiso 6A). Ukungezelela, amatjhuguluko amakhulu abonakalako, khulukhulu i-helix de (i-helix emfitjhani phakathi kwe-TMH D ne-E) lapho i-L-Phe217 ithuthelwa khona ephaketheni lokubopha le-QB kanye nokujikeleziswa kwe-L-Tyr223 (Umfanekiso 6A) Ukuphula ibhondi ye-hydrogen nama-M-Ap4 begodu kuvale uhlaka lokubopha kwe-QB isayithi (Umfanekiso 6B). I-Helix de pivots esisekelweni sayo, i-Cα ye-L-Ser209 itjhuguluka nge-0.33Å, kuthi i-L-Val221Cα itjhugululwe nge-3.52Å. Akukho ukutjhuguluka okubonwako ku-TMH D no-E, okuhlanganiswa kizo zombili izakhiwo (Umfanekiso 6A). Ngokwazi kwethu, lesi sisakhiwo sokuthoma ku-RC yemvelo esivala indawo ye-QB. Ukumadanisa nesakhiwo esipheleleko (esibophelwe ku-QB) kutjengisa bona ngaphambi kobana i-quinone yehliswe, kutlhogeka ukutjhuguluka kwe-conformational ukwenza bona ingene ku-quinone. I-L-Phe217 iyajikeleza ukwakha ukusebenzisana kwe-π-stacking nesiqhema sehloko se-quinone, begodu i-helix iyatjhugulukela ngaphandle, ivumela ithambo le-L-Gly222 kanye ne-side chain ye-L-Tyr223 ukwakha inethiwekhi ye-hydrogen bond enesakhiwo se-hydrogen bond esizinzileko (Umfanekiso 6, A no-C).
(A) Ikhathuni edlulako yehologramu (i-L chain, i-orange/M chain, i-magenta) kanye nesakhiwo se-apo (grey), lapho iinsalela eziqakathekileko zitjengiswa ngendlela yokujanyelwa okufana nenduku. I-UQ10 imelelwa yibha ephuzi. Umugqa onamachashazi utjengisa amabhondi we-hydrogen akhiwe kiyo yoke isakhiwo. (B no-C) Ukuvezwa kwe-apolipoprotein kanye nesakhiwo sendandatho yoke, okuveza i-oksijini ye-side chain ye-L-Phe217 ngombala oluhlaza okwesibhakabhaka kanye ne-L-Tyr223 ngombala obomvu, ngokulandelana. Iyunithi ye-L i-orentji; amayunithi we-M ne-H awafakwa imibala. (D no-E) I-Apolipoprotein (D) kanye nendawo yoke (E) ye-RC QB [umbala ngu-(A) ngokulandelana] kanye ne-Thermophilus thermophilus PSII (okuluhlaza, ohlaza satjani nge-quinone yeplastiki; i-PDB ID: 3WU2) Hlanganisa (58).
Ngokungalindelekileko, nanyana izakhiwo ezinengi ze-QB-deficient RCs ngaphandle kwe-LH1 zitholakala, amatjhuguluko we-conformational abonwe esifundweni lesi azange abikwe ngaphambilini. Lokhu kufaka hlangana isakhiwo sokuqedwa kwe-QB esivela ku-Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) kanye ne-Rba. sphaeroides (PDB ID: 1OGV) (29), zoke zifana nesakhiwo sazo se-QB. Ukuhlolisisa i-3PRC kuveze bona ama-molecule we-LDAO (Lauryl Dimethyl Amine Oxide) ahlangana emnyango wendawo ye-QB, okungavimbela ukuhleleka kabutjha kube yindlela evalekileko. Nanyana i-LDAO ingaboli endaweni efanako ku-1EYS nofana ku-1OGV, ama-RC la alungiselelwe kusetjenziswa isisetjenziswa esifanako begodu angakhiqiza umphumela ofanako. Isakhiwo sekristalu se-Rba. I-Sphaeroides RC ehlangene ne-cytochrome c2 (PDB ID: 1L9B) nayo ibonakala inendawo ye-QB evaliweko. Kodwana, endabeni le, indawo ye-N-terminal ye-RC-M polypeptide (esebenzisana nendawo yokubophelela ye-QB nge-H bond ye-Tyr residue ku-Q helix) yamukela ukubunjwa okungasikho kwemvelo, begodu ukutjhuguluka kokwakheka kwe-QB akuhlolwa godu (30 ). Okuqinisekisako kukuthi asikaboni ukutjhuguluka okunje kwe-M polypeptide esakhiweni se-RC-LH114-W, okupheze kufane nendawo ye-N-terminal ye-RC-LH116 RC. Kufanele kuqashelwe nokuthi ngemva kokuqedwa kwe-antenna ye-LH1 esekelwe ku-detergent, ama-apolipoprotein RCs ku-PDB ararululwe, okwaqeda amadamu wangaphakathi we-quinone nama-lipids esikhaleni phakathi kwe-RC kanye nendawo yangaphakathi ye-LH1 ring ezungezeko (31, 32). I-RC ihlala isebenza ngombana igcina woke ama-cofactors, ngaphandle kwe-QB quinone ebolako, engazinzileko begodu evamise ukulahleka ngesikhathi sokulungiselela (33). Ukungezelela, kuyaziwa bona ukususwa kwe-LH1 kanye namafutha we-cyclic wemvelo ku-RC kungaba nomthelela emisebenzini, njengokuphila okufitjhani kwe-P+QB-state ehlukaniswe nge-charge (31, 34, 35). Ngalokho-ke, siqagela bona ukuba khona kwendandatho ye-LH1 yendawo ezungeze i-RC kungagcina indawo ye-QB “evaliweko”, ngaleyo ndlela kulondoloze ibhoduluko lendawo eduze ne-QB.
Nanyana i-apolipoprotein (ngaphandle kwe-QB quinone) kanye nesakhiwo esipheleleko simele iinthombe ezimbili kwaphela zokutjhuguluka kwendawo ye-QB, kunokobana kube mlandelande wezehlakalo, kuneenkomba zokuthi ukubopha kungavalwa ukukhandela ukubopha kabutjha nge-hydroquinone Ukukhandela ukuvinjelwa kwe-substrate. Ukusebenzisana kwe-quinolol ne-quinone eduze nendawo ye-QB ye-apolipoprotein kungaba ngokuhlukileko, okurholela ekulahlweni kwayo yi-RC. Sekuyisikhathi eside kuphakanyiswa bona amatjhuguluko wobujamo adlala indima ekubopheni nasekwehliseni ama-quinones. Ikhono lama-RC aqhwaziweko lokwehlisa ama-quinones ngemva kokutjhuguluka kobumnyama liyakhubazeka (36); I-X-ray crystallography itjengisa bona umonakalo lo ubangelwa kukuthi ama-QB quinones avaleleke "kude" okuma-4.5 Å ukusuka endaweni esebenzako eseduze (26) , 37). Siphakamisa bona ukubophelela okukude lokhu kusithombe sesimo esiphakathi phakathi kwe-apolipoprotein kanye nesakhiwo sendandatho epheleleko, okulandela ukusebenzisana kokuthoma ne-quinone nokuvulwa kwendawo ye-QB.
Umhlobo we-II RC otholakala ku-PSII complex yamabhaktheriya athile we-phototrophic kanye nama-cyanobacteria, i-algae kanye neentjalo inokwakheka nokusebenza kokulondolozwa (38). Ukulungiswa kwesakhiwo okutjengiswe kuMfanekiso 6 (D no-E) kugandelela ukufana hlangana nama-PSII RCs kanye nendawo ye-QB ye-RC yebhaktheriya. Ukumadanisa lokhu kade kuyisibonelo sokufunda amahlelo ahlobene khulu wokubopha nokunciphisa kwe-quinone. Iincwadi zangaphambilini ziphakamise bona amatjhuguluko we-conformational akhambisana nokwehliswa kwe-PSII yama-quinones (39, 40). Ngalokho-ke, nakuqalwa ukulondolozwa kwe-RC, indlela yokubopha engakaqalwa ngaphambilini ingasebenza nesayithi ye-QB ye-PSII RC eentjalweni ezine-oksijini ezine-phototrophic.
I-Rps ΔpufW (unlabeled pufW deletion) kanye ne-PufW-His (C-terminal 10x His-tagged protein-W expressed from natural pufW locus) strains. i-palustris CGA009 yachazwa emsebenzini wethu wangaphambilini (16). Iinhlobo lezi kanye nomzali we-isogenic wild-type zatholwa efrijini ngokufaka inani elincani lamaseli ku-PYE (i-5 g litre ngayinye -1) (egcinwe ku-LB ku--80 °C, equkethe i-50% ( w/v) glycerol) iphrotheyini, i-yeast extract kanye ne-succinate) agar [1.5%/v) ipuleti. Ipuleti lafukanywa ubusuku boke ebumnyameni emazingeni womtjhiso wegumbi ngaphasi kweemeko ezinganamoya, bese lakhanyiswa ngokukhanya okumhlophe (~50 μmolm-2 s-1) okunikelwe ngama-OSRAM 116-W halogen bulbs (RS Components, UK) amalanga ama-3 ukuya kwama-5 bekwaba kulapho kuvela khona ikoloni yinye. Ikoloni eyodwa yasetjenziselwa ukugoma i-10 ml ye-M22+ medium (41) efakwe i-0.1% (w/v) yama-casamino acid (lapha ebizwa ngokuthi yi-M22). Isiko lakhuliswa ngaphasi kweemeko ezine-oksijini ephasi ebumnyameni ku-34°C ngokunyakaza nge-180 rpm ama-awa ama-48, bese kuthi i-70 ml yesiko yafakwa ngaphasi kweemeko ezifanako ama-awa ama-24. I-semi-aerobic culture enomthamo we-1 ml isetjenziselwa ukugoma i-30 ml ye-M22 medium ngaphakathi kwebhodlela lengilazi elikhanyako elingama-30 ml begodu lifakwe imisebe ngokutjhukumisa (~50μmolm-2 s-1) ama-awa ama-48 ngamandla kazibuthe ahlanzekileko. Bese kuthi i-30 ml yesiko yagonywa ngelitha elilodwa lesintu ngaphasi kweemeko ezifanako, ebegade isetjenziselwa ukugonywa ngamalitha angaba li-9 wesintu esikhanyiswe ku-~200 μmolm-2 s-1 ama-awa ama-72. Amaseli avunwa ngokusebenzisa i-centrifugation ku-7132 RCF imizuzu ema-30, abuyiselwa emuva ku-~10 ml ye-20 mM tris-HCl (pH 8.0), begodu agcinwa ku--20°C bekwaba kulapho atlhogeka khona.
Ngemva kokuncibilika, faka amanye amakristalu we-deoxyribonuclease I (Merck, UK), i-lysozyme (Merck, UK) kanye namaphilisi amabili we-Roche holoenzyme protease inhibitor (Merck, UK) kumaseli ahlelwe kabutjha. Ngaphakathi kwe-20,000 psi French pressure cell (Aminco, USA), amaseli aphazanyiswa amahlandla ayi-8 ukuya kwayi-12. Ngemva kokususa amaseli angaphukileko kanye neemvuthuluka ezingancibilikiko ngokutjhugulula ku-18,500 RCF imizuzu eli-15 ku-4°C, ulwelwesi lwakhishwa kusuka ku-lysate enebala ngokutjhugululwa ku-113,000 RCF ama-awa ama-2 ku-43,000°C. Lahla ingcenye encibilikako bese ubuyisela i-membrane enombala ku-100 ukuya ku-200 ml ye-20 mM tris-HCl (pH 8.0) bese uyihlanganisa bekube kulapho kungabi nama-aggregates abonakalako. I-membrane elengisiweko yafukanywa ku-20 mM tris-HCl (pH 8.0) (Anatrace, USA) ene-2% (w/v) β-DDM i-awa elilodwa ebumnyameni ku-4°C ngokuvuselelwa okuncani. Bese u-centrifuge ku-70°C ukuncibilikisa i-150,000 RCF ku-4°C nge-awa elilodwa ukususa izinto ezingancibilikiko eziseleko.
I-solubilizing membrane evela ku-ΔpufW strain yasetjenziswa ku-50 ml DEAE Sepharose ion exchange column ene-3 column volumes (CV) ye-binding buffer [20 mM tris-HCl (pH 8.0) equkethe i-0.03% (w / v) β-DDM]. Hlamba ikholomu ngamabhafa amabili wokubopha we-CV, bese ugeza ikholomu ngamabhafa amabili wokubopha ane-50 mM NaCl. I-RC-LH116 complex yakhishwa nge-linear gradient ye-150 ukuya ku-300 mM NaCl (ku-binding buffer) ku-1.75 CV, begodu i-binding complex eseleko yakhishwa nge-binding buffer ene-300 mM NaCl ku-0.5 CV. Buthelela i-absorption spectrum phakathi kuka-250 no-1000 nm, gcina ingcenye ene-absorbance ratio (A880/A280) engaphezu kwe-1 ku-880 ukuya ku-280 nm, uyihlambulule kabili ku-binding buffer, bese usebenzisa ikambiso efanako godu kukholomu ye-DEAE On purification. Nciphisa amafraktjhini ane-A880/A280 ephezulu kune-1.7 kanye ne-A880/A805 ephezulu kune-3.0, yenza umjikelezo wesithathu wokutjhentjhana kwama-ayoni, begodu ugcine amafraktjhini anama-A880/A280 aphezulu kune-2.2 kanye nama-A880/A805 aphezulu kune-5.0.0. I-complex ehlanjululwe kancani yahlanganiswa yaba yi-~2 ml ku-Amicon 100,000 molecular weight cut-off (MWCO) centrifugal filter (Merck, UK), begodu yafakwa ku-Superdex 200 16/600 size exclusion column (GE Healthcare, US) ene-200 mM ye-NaCl, bese yafakwa ku-buffer efanako ku-15. I-CV. Buthelela ama-absorption spectra we-sayizi exclusion fraction, bese ugxilisa ama-absorption spectra nge-A880/A280 ratio ngaphezulu kwe-2.4 kanye nama-A880/A805 ratio ngaphezulu kwe-5.8 ukuya ku-100 A880, bese uwasebenzisela msinyana ukulungiselela igridi ye-cryo-TEM nofana ukubulunga Gcina ku-80°C bekube kulapho kutlhogeka khona.
I-solubilizing membrane evela ku-PufW-His strain yasetjenziswa ku-20 ml HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) ene-200 mM NaCl kanye ne-0.03% (w/w)) ku-IMAC buffer ( GE Healthcare). v) β-DDM]. Ikholomu yahlanzwa ngama-CV amahlanu we-IMAC buffer, bese kuthi ngama-CV amahlanu we-IMAC buffer ene-10 mM histidine. I-core complex yasuswa ekholomu ngamabhafa amahlanu we-IMAC ane-100 mM histidine. Ingcenye equkethe i-RC-LH114-W complex ihlanganiswe ibe yi-~10 ml ethangini elivuthiweko elihlonywe nge-Amicon 100,000 MWCO filter (Merck, UK), lihlanjululwe amahlandla ama-20 nge-binding buffer, bese lifakwa ku-25 ml Kukholomu ye-DEAE Sepharose, ama-CV amane asetjenziswa ngaphambilini ku-vava. Hlamba ikholomu ngamabhafa amane wokubophelela ama-CV, bese ukhipha i-complex kuma-CV abunane nge-linear gradient ye-0 ukuya ku-100 mM NaCl (nge-binding buffer), begodu ama-CV amane aseleko ane-100 mM binding buffer Ama-complex asele ahlutjhwe ku-sodium chloride ahlanganiswe ne-A8880/880/A2000 kanye nesilinganiso esiphezulu kune-A2. Isilinganiso se-A880/A805 esingaphezu kwamafraktjhini ama-4.6 sahlanganiswa ukuya ku-~2 ml ku-Amicon 100,000 MWCO centrifugal filter, begodu sazaliswa nge-1.5 CV IMAC ngaphambilini. Buthelela ama-absorption spectra wamafraktjhini wokukhipha ubukhulu bese ugxilisa ama-absorption spectra nge-A880/A280 ama-ratio ngaphezulu kwe-2.1 kanye nama-A880/A805 ama-ratio angaphezu kwama-4.6 ukuya ku-100 A880, asetjenziselwa khonokho ukulungiselela igridi ye-TEM efriziweko nofana agcinwe ku-80°C bekube lapho kutlhogeka khona.
Ifriji ye-Leica EM GP yasetjenziselwa ukulungiselela amagridi we-TEM wezinga lokutjhisa eliphasi. I-complex yahlanjululwa ku-IMAC buffer ukuya ku-A880 ka-50, bese i-5μl yafakwa ku-QUANTIFOIL 1.2/1.3 carbon-coated copper mesh (Agar Scientific, UK). Fukamela igridi ku-20°C kanye no-60% womswakama ohlobene ama-30 s, bese uyisula yome ama-3 s, bese uyicima nge-ethane ewuketshezi ku--176°C.
Idatha ye-RC-LH114-W complex yarekhodwa ku-eBIC (Electronic Bioimaging Center) (I-British Diamond Light Source) nge-Titan Krios microscope, esebenza nge-accelerating voltage ye-300kV, nge-nominal magnification ye-130,000× kanye namandla we-choose gap ye-20V. I-Gatan 968 GIF Quantum ene-K2 peak detector yasetjenziselwa ukurekhoda iinthombe ngendlela yokubala ukubuthelela idatha. Ubukhulu bephikseli obulinganisiweko ngu-1.048Å, begodu izinga lomthamo ngu-3.83 e-Å-2s-1. Siqoqe imuvi ngemizuzwana eli-11 bese siyihlukanisa yaba ziingcenye ezima-40. Sebenzisa indawo enekhabhoni ukugxila kabutjha isibonakhulu, bese uqoqa amamuvi amathathu ngomgodi ngamunye. Sekukonke, amamuvi ama-3130 abuthelelwa, ngamanani wokungagxili hlangana no--1 no--3μm.
Idatha ye-RC-LH116 complex yabuthelelwa kusetjenziswa i-microscope efanako e-Asterbury Biostructure Laboratory (University of Leeds, UK). Idatha yabuthelelwa ngendlela yokubala ngokukhuliswa kwe-130 k, begodu ubukhulu bephikseli balinganiswa ku-1.065 Å ngomthamo we-4.6 e-Å-2s-1. Ifilimu yarekhodwa ngemizuzwana eli-12 begodu yahlukaniswa yaba ziingcenye ezima-48. Sekukonke, amafilimu ama-3359 aqoqwe, ngamanani wokungagxili phakathi kuka--1 no--3μm.
Konke ukucutshungulwa kwedatha kwenziwa ngephayiphi ye-Relion 3.0 (42). Sebenzisa i-Motioncorr 2 (43) ukulungisa ukunyakaza kwe-beam ngokulinganisa umthamo, bese usebenzisa i-CTFFIND 4.1 (44) ukunquma ipharamitha ye-CTF (umsebenzi wokudlulisa ukumadanisa). Iinthombe ezijayelekileko ngemva kwamagadango wokuthoma wokucubungula zitjengiswe kuMfanekiso 2. S16. Isifanekiso sokukhetha esizenzakalelayo senziwa ngokukhetha ngesandla amaphikseli angaba ma-250 weenhlayiya ezili-1000 kufreyimu yamaphikseli ama-250 begodu kungabi nokuhlukaniswa kwemikhakha emibili (2D), ngaleyo ndlela kulahlwe lezo zihlukaniso ezihlangabezana nokusilaphazeka kwesampula nofana ezingenazimpawu ezibonakalako. Bese, ukukhetha okuzenzakalelayo kwenziwa kizo zoke iinthombe ezincani, begodu i-RC-LH114-W beyingama-particle angu-849,359, begodu i-RC-LH116 beyingama-particle angu-476,547. Zoke iinhlayiya ezikhethiweko zidlule emizuliswaneni emibili yokuhlukaniswa okungasikho kwe-2D, begodu ngemva kokugijima ngamunye, iinhlayiya ezihlangabezana nendawo yekhabhoni, ukusilaphazeka kwesampula, akukho zimpawu ezibonakalako nofana iinhlayiya ezidlula khulu ziyalahlwa, okurholela ekutheni kusetjenziswe ama-particle angama-772,033 (90.9%) kanye nama-359,678 (75.55) wokuhlukaniswa kwe-3D. I-RC-LH114-W kanye ne-RC-LH116 ngokulandelana. Imodeli yokuthoma ye-3D yenziwa kusetjenziswa indlela ye-stochastic gradient descent. Ukusebenzisa imodeli yokuthoma njengesikhombo, iinhlayiya ezikhethiweko zihlukaniswe zaba ziinkoro ezine ku-3D. Ukusebenzisa imodeli yesigaba lesi njengesikhombo, yenza ukucwengisiswa kwe-3D eenhlayiyani ezisesigabeni esikhulu, bese usebenzisa isihlungi sokuthoma se-15Å sokudlula phasi ukuvala indawo ye-solvent, engeza amaphikseli ayi-6 wamaphethelo athambileko, bese usebenza ngemva kokucubungula amaphikseli ukulungisa umsebenzi wokudlulisa i-Gatan K2 peak Modulation we-top detector. Ngedathasethi ye-RC-LH114-W, imodeli yokuthoma le yatjhugululwa ngokususa ukuminyana okuqinileko emaphethelweni wemaski (okungahlanganiswa nokuminyana okuyinkimbinkimbi okuyinhloko ku-UCSF Chimera). Amamodeli aphumelako (iinqunto ze-RC-LH114-W ne-RC-LH116 ziyi-3.91 ne-4.16 Å, ngokulandelana) asetjenziswa njengesikhombo somzuliswano wesibili wokuhlukaniswa kwe-3D. Iinhlayiya ezisetjenzisiweko zihlanganiswe zibe sesigabeni sokuthoma se-3D begodu azinakho ukuhlobana okuqinileko nendawo. Ukudlulana nofana ukutlhogeka kweempawu ezibonakalako zesakhiwo. Ngemva komjikelezo wesibili wokuhlukaniswa kwe-3D, kukhethwe isigaba esine-resolution ephezulu [Ku-RC-LH114-W, isigaba sinye ngama-particles angama-377,703 (44.5%), ku-RC-LH116, kunemikhakha emibili, ehlanganisa ama-particles angama-260,752 (54.7% Wh), lokha nasele ahlelwe ngemva kwawo afanako kwaphela. ukujikeleza kokuthoma ngomehluko omncani]. Iinhlayiya ezikhethiweko zikhishwa kabutjha ebhokisini lamaphikseli ama-400 bese zicwengiswa nge-3D. Imaski ye-solvent yenziwa kusetjenziswa isihlungi sokuthoma se-15Å, ukunwetshwa kwemephu yamaphikseli ama-3 kanye nemaski ethambileko yamaphikseli ama-3. Ukusebenzisa ukucwengisiswa kwe-CTF nge-particle ngayinye, ukulungiswa kokunyakaza kwe-particle ngayinye kanye nomjikelezo wesibili wokucwengisiswa kwe-CTF nge-particle ngayinye, ukucwengisiswa kwe-3D, ukufihla i-solvent nokucubungula ngemva kwesinyathelo kwenziwa ngemva kwesinyathelo ngasinye ukuthuthukisa ukuthungwa okuphumelako. Ngokusebenzisa i-FSC (Fourier Shell Correlation Coefficient) inani lokusika le-0.143, iinqunto zamamodeli wokugcina we-RC-LH114-W kanye ne-RC-LH116 ziyi-2.65 ne-2.80Å, ngokulandelana. Ijika le-FSC lemodeli yokugcina litjengiswe kuMfanekiso 2. S17.
Zoke iimlandelande zamaphrotheyini zilandwa 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); Iphrotheyini-W (PufW; UniProt ID: Q6N1K3). I-SWISS-MODEL (45) yasetjenziselwa ukwakha imodeli ye-homology ye-RC, equkethe ukulandelana kwamaphrotheyini we-RC-L, RC-M kanye ne-RC-H kanye nesakhiwo sekristalu se-Rba. i-sphaeroides RC yasetjenziswa njengethempulethi (PDB ID: 5LSE) (46). Sebenzisa ithuluzi le-“fit map” ku-UCSF Chimera ukulinganisa imodeli ekhiqiziweko kumephu (47), uthuthukise isakhiwo samaphrotheyini, begodu u-cofactor [4×BChl a (ibizo le-monomer library residue name = BCL), 2×BPh a (BPH), umhlobo owodwa nofana emibili ye-UQ10 (U10), i-non-heme iron eyodwa (Fe) kanye ne-hexa-hexadi, 44 (QAK)] sebenzisa i-Coot (48) ukufaka. Njengombana i-QAK ingatholakali elayibhrari ye-monomer, yafakwa ipharamitha ngokusebenzisa ithuluzi le-eLBOW ku-PHENIX (49).
Okulandelako, iyunithi ye-LH1 yakhiwa. Ekuthomeni, ithulusi lokwakha elizenzakalelayo ku-PHENIX (49) lasetjenziselwa ukwakha ngokuzenzakalelako ingcenye yokulandelana kwe-LH1 kusetjenziswa imephu kanye nokulandelana kwamaphrotheyini we-LH1-α ne-LH1-β njengokufaka. Khetha i-LH1 epheleleko, uyikhuphe bese uyifaka ku-Coot, ufake ngesandla ukulandelana okulahlekileko kiyo, bese ucwengisisa ngesandla isakhiwo soke ngaphambi kokufaka ama-BCls amabili a (BCL) kanye ne-spirilloxanthin (CRT) [ngokuya nge-Rps efaneleko Ukuminyana kwe-LH1 complex kanye nokuqukethwe kwe-carotenoid eyaziwako. Imihlobo (17)]. Kopa iyunithi epheleleko ye-LH1, bese usebenzisa i-UCSF Chimera “Ithulusi leMephu yokuDoba” ukudosa endaweni eseduze engasi yimodeli ye-LH1, bese uyicwengisisa ku-Coot; phinda ikambiso bekube lapho woke ama-subunits we-LH1 sele amodelwe. Ngesakhiwo se-RC-LH114-W, ngokukhipha ukuminyana okungakabiwa ku-Coot, iphrotheyini ihlukaniswa eengcenyeni eziseleko ezingezizo zamaphrotheyini kumephu ye-USCF Chimera begodu ithulusi lokwakha i-Autobuild lisetjenziselwa ukusungula imodeli yokuthoma, kanye nama-subunits aseleko (iphrotheyini-W) Ukumodela. E-PHENIX (49). Faka nanyana ngikuphi ukulandelana okulahlekileko kumodeli ephumelako ku-Coot (48), bese ucwengisisa ngesandla yoke iyunithi. Ukuminyana okungakabikwa okuseleko kufanelana nokuhlanganiswa kwamafutha (PDB monomer library ID of CDL = CDL, POPC = 6PL kanye ne-POPG = PGT), i-β-DDM detergent (LMT) kanye nama-molecules we-UQ10 (U10). Sebenzisa ukuthuthukiswa kwe-PHENIX (49) nokulungiswa ngesandla ku-Coot (48) ukuphelelisa imodeli yokuthoma epheleleko bekube kulapho iimbalo zemodeli kanye nekhwalithi ebonakalako yokufaneleka kungathuthukiswa godu. Ekugcineni, sebenzisa i-LocScale (50) ukulola imephu yendawo, bese wenza eminye imijikelezo eminengi yokumodela ukuminyana okungakabiwa kanye nokwenza ngcono okuzenzakalelayo nangokuzenzakalelayo.
Ama-peptides, ama-cofactors namanye ama-lipids nama-quinones afakwe ngaphakathi kwama-density wawo atjengiswe ku-Figure 1 no-2. S18 ukuya ku-S23. Ilwazi leembalo lemodeli yokugcina litjengiswe kuThebuli S1.
Ngaphandle kobana kuvezwe ngenye indlela, ama-UV/Vis/NIR absorption spectra abuthelelwa ku-Cary60 spectrophotometer (Agilent, USA) ngeenkhathi eziyi-1 nm ukusuka ku-250 nm ukuya ku-1000 nm kanye nesikhathi sokuhlanganiswa esingu-0.1s.
Nciphisa isampula nge-quartz cuvette enendlela eyi-2 mm eya ku-A880 ye-1, bese uqoqa i-absorption spectrum hlangana ne-400 ne-1000 nm. I-circular dichroic spectra yaqoqwa ku-Jasco 810 spectropolarimeter (Jasco, Japan) nge-1 nm phakathi kwe-400 nm kanye ne-950 nm ngezinga lokuskena le-20 nm min-1.
I-molar extinction coefficient inqunywa ngokuncibilikisa i-core complex ibe yi-A880 engaba ngu-50. Nciphisa ivolumu ye-10μl ku-990μl ye-binding buffer nofana i-methanol, bese uqoqa i-absorption spectrum khonokho ukunciphisa ukuwohloka kwe-BChl. Okuqukethwe kwe-BChl yesampula ngayinye ye-methanol kwabalwa nge-extinction coefficient ku-771 nm ye-54.8 mM-1 cm-1, begodu i-extinction coefficient yatholakala (51). Hlukanisa ukugxila kwe-BChl okulinganisiweko ngo-32 (RC-LH114-W) nofana ngo-36 (RC-LH116) ukuthola ukugxila kwe-core complex, okuzokusetjenziswa ukuthola i-absorption spectrum yesampula efanako ebuthelelwe ku-buffer Extinction coefficient. malungana. Kwathathwa iinlinganiso ezintathu eziphindaphindiweko kusampula ngayinye, begodu ukumuncwa okuphakathi kwe-BChl Qy okuphezulu kwasetjenziselwa ukubala. I-extinction coefficient ye-RC-LH114-W elinganiswa ku-878 nm ngu-3280±140 mM-1 cm-1, ngesikhathi i-extinction coefficient ye-RC-LH116 elinganiswa ku-880 nm ngu-3800±30 mM-1 cm-1.
I-UQ10 yalinganiswa ngokuya ngendlela eku (52). Ngamafuphi, i-HPLC yesigaba esibuyela emuva (RP-HPLC) yenziwa kusetjenziswa ihlelo le-Agilent 1200 HPLC. Ncibilikisa i-0.02 nmol ye-RC-LH116 nofana i-RC-LH114-W ku-50μl ye-50:50 methanol:chloroform ene-0.02% (w/v) ye-ferric chloride, bese ujova i-Beckman Coulter Ultrasphere ODS 4 elinganiselwe ngaphambilini Ncibilikisa ku-1-1-m. 40°C ku-HPLC solvent (80:20 methanol:2-propanol) kukholomu eyi-×25 cm. Yenza i-isocratic elution ku-HPLC solvent ukutjheja ukumuncwa ku-275 nm (UQ10), 450 nm (carotenoids) kanye no-780 nm (BChl) nge-awa elilodwa. I-peak ku-275 nm chromatogram emizuzwini ema-25.5 yahlanganiswa, ebeyinganawo amanye amakhompawundi abonakalako. Indawo ehlanganisiweko isetjenziselwa ukubala inani le-molar le-UQ10 elikhutjhwe ngokuya ngejika lokulinganisa elibalwe ukusuka ekujovweni kwamazinga ahlanzekileko ukusuka ku-0 ukuya ku-5.8 nmol (Umfanekiso S14). Isampula ngayinye yahlaziywa kathathu, begodu iphutha elibikweko lihambisana ne-SD yesilinganiso.
Isixazululo esiqukethe i-RC-LH1 complex ene-Qy ephezulu yokumuncwa kwe-0.1 salungiswa nge-30 μM eyehlisiwe ye-horse heart cytochrome c2 (Merck, UK) kanye ne-0 ukuya ku-50 μMUQ2 (Merck, UK). Amasampula amathathu we-1-ml alungiselelwe nge-UQ2 ngayinye begodu afukamela ubusuku boke ebumnyameni ku-4°C ukuqinisekisa ukutjhuguluka okupheleleko ebumnyameni ngaphambi kokulinganisa. Isisombululo safakwa ku-OLIS RSM1000 modular spectrophotometer ene-300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm middle kanye 0.6 mm outlet slits. I-600 nm ende pass filter ibekwa emnyango wesampula ye-phototube kanye ne-reference photomultiplier tube ukukhipha ukukhanya kokuvusa. Ukumuncwa kwagadwa ku-550 nm ngesikhathi sokuhlanganiswa esingu-0.15 s. Ukukhanya kokuvusa kukhishwa ku-880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) ngekheyibuli ye-fiber optic nge-90% yamandla nge-DC2200 controller (Thorlabs Ltd., UK) begodu kukhishwa emthonjeni wokukhanya nge-engeli ye-90°. Umsebe wokulinganisa ujamelene nesibuko ukubuyisela nanyana ngikuphi ukukhanya ebekungamunwa yisampula ekuthomeni. Qala ukumuncwa kwe-10 s ngaphambi kokukhanya kwe-50 s. Bese ukumuncwa kwaragela phambili nokugadwa ama-60 s ebumnyameni ukuhlola izinga lapho i-quinolol yehlisa khona i-cytochrome c23 + (qala isithombe S8 malungana nedatha engakalungiswa).
Idatha yasetjenzwa ngokufaka izinga lokuthoma eliqondileko ngaphakathi kwe-0.5 ukuya ku-10 s (kuya ngobukhulu be-UQ2) nokulinganisa amazinga wamasampula amathathu kiwo woke amazinga we-UQ2. Ukugxila kwe-RC-LH1 okubalwe nge-extinction coefficient ehlukileko kwasetjenziselwa ukutjhugulula izinga libe yi-catalytic efficiency, kwafakwa ku-Origin Pro 2019 (OriginLab, USA), begodu kwafakwa ku-Michaelis-Menten model ukuthola amanani we-Km ne-Kcat.
Ukulinganisa ukumuncwa kwesikhatjhana, isampula ye-RC-LH1 yahlanjululwa yaba yi-~2μM ku-IMAC buffer ene-50 mM ye-sodium ascorbate (Merck, USA) kanye ne-0.4 mM Terbutin (Merck, USA). I-ascorbic acid isetjenziswa njengomnikeli we-elekthronikhi, begodu i-tert-butaclofen isetjenziswa njengesivimbeli se-QB ukuqinisekisa bona umnikeli omkhulu we-RC uhlala anciphisiwe (okutjho bona, angasi nge-photooxidized) kiyo yoke ikambiso yokulinganisa. Pheze i-3 ml yesampula ifakwa eselini elijikajikako (elilinganiselwa ku-0.1 m ngobubanzi, 350 RPM) elinobude bendlela yokukhanya eyi-2 mm ukuqinisekisa bona isampula endleleni ye-laser inesikhathi esaneleko sokuzivumelanisa nobumnyama hlangana nama-pulse wokuvusa. Sebenzisa ama-~100-fs laser pulses ukukhulisa i-Ti: Sapphire laser system (Spectra Physics, USA) ukuvusa isampula ku-880 nm ngezinga lokuphindaphinda le-1 kHz (20 nJ ye-NIR nofana i-100 nJ ye-Vis). Ngaphambi kokubuthelela idatha, veza isampula ekukhanyeni okuvuthako isikhathi esingaba mizuzu ema-30. Ukuvezwa kuzokubangela ukungasebenzi kwe-QA (mhlamunye ukwehlisa i-QA kanye nofana kabili). Kodwana sibawa uqaphele bona ikambiso le iyabuyiselwa emuva ngombana ngemva kwesikhathi eside sokutjhuguluka ebumnyameni, i-RC izokubuyela kancanikancani emsebenzini we-QA. I-Helios spectrometer (Ultrafast Systems, USA) yasetjenziselwa ukulinganisa i-transient spectra ngesikhathi sokulibaziseka esingu--10 ukuya ku-7000 ps. Sebenzisa isofthiwe ye-Surface Xplorer (Ultrafast Systems, USA) ukuhlukanisa amasethi wedatha, bese uhlanganisa begodu uwalinganise. Sebenzisa iphakheji yesofthiwe ye-CarpetView (Light Conversion Ltd., Lithuania) ukusebenzisa isethi yedatha ehlanganisiweko ukuthola ama-spectra ahlukileko ahlobene nokubola, nofana usebenzise umsebenzi ohlanganisa ama-exponent amanengi ngempendulo yesisetjenziswa ukulingana nokutjhuguluka kwe-single-wavelength spectral ku-Origin (OriginLab, USA).
Njengombana kutjhiwo ngehla (53), kwalungiswa ifilimu ye-photosynthetic ene-LH1 complex engenazo zombili i-RC ne-peripheral LH2 antenna. I-membrane yahlanjululwa nge-20 mM tris (pH 8.0) bese yafakwa ngaphakathi kwe-quartz cuvette ene-2 ​​mm optical path. I-30nJ laser pulse yasetjenziselwa ukuvusa isampula ku-540 nm ngesikhathi sokulibaziseka se--10 ukuya ku-7000 ps. Sebenzisa isethi yedatha njengombana kuchaziwe nge-Rps. isampula yomngani.
I-membrane yafakwa nge-centrifugation ku-150,000 RCF ama-awa ama-2 ku-4°C, bese ukumuncwa kwayo ku-880 nm kwabuyiselwa emuva ku-20 mM tris-HCl (pH 8.0) kanye ne-200 mM NaCl. Ncibilikisa i-membrane ngokuyivuselela kancanikancani ku-2% (w/v) β-DDM i-awa elilodwa ebumnyameni ku-4°C. Isampula yahlanjululwa ku-100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) ukuya ekugxileni kwamaphrotheyini we-2.5 mg ml-1 (ukuhlaziywa kwe-Bio-Rad). Ukusetjenziswa okuragela phambili kwenziwa ngendlela egadangiswe ngaphambilini (54), kuthoma ngokunciphisa amaphrotheyini ama-50 μg abe yi-50 μl ye-TEAB ene-1% (w/v) ye-sodium laurate (Merck, UK). Ngemva kokuzwakala kwamasekondi ama-60, yehliswa nge-5 mM tris(2-carboxyethyl)phosphine (Merck, UK) ku-37°C imizuzu ema-30. Ukwenza i-S-alkylation, faka isampula nge-10 mM methyl S-methylthiomethanesulfonate (Merck, UK) bese uyifaka ukusuka ku-200 mM isopropanol stock solution imizuzu eli-10 emazingeni womtjhiso wegumbi. Ukugaya kwamaphrotheyini kwenziwa ngokufaka i-2 μg ye-trypsin/endoproteinase Lys-C mixture (Promega UK) begodu kwafukanywa ku-37°C ama-awa ama-3. I-laurate surfactant yakhishwa ngokufaka i-50 μl ye-ethyl acetate kanye ne-10 μl ye-10% (v/v) ye-LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) bese i-vortexing ama-60 s. Ukuhlukaniswa kwesigaba kwakhuthazwa ngokutjhugulula ku-15,700 RCF imizuzu emi-5. Ngokuya kwephrothokholi yomkhiqizi, i-C18 spin column (i-Thermo Fisher Scientific, UK) yasetjenziselwa ukudonsa ngokutjheja nokususa itswayi esigabeni esingezansi esine-peptide. Ngemva kokomisa nge-vacuum centrifugation, isampula yancibilikiswa ku-0.5% TFA kanye ne-3% acetonitrile, begodu i-500 ng yahlaziywa nge-nanoflow RP chromatography ehlanganiswe ne-mass spectrometry kusetjenziswa amapharamitha wehlelo achazwe ngaphambilini.
Sebenzisa i-MaxQuant v.1.5.3.30 (56) ukuthola amaphrotheyini nokulinganisa ukufuna ama-Rps. idathabheyisi ye-palustris proteome (www.uniprot.org/proteomes/UP000001426). Idatha ye-mass spectrometry proteomics ifakwe ku-ProteomeXchange Alliance nge-PRIDE partner repository (http://proteomecentral.proteomexchange.org) ngaphasi kwe-dataset identifier PXD020402.
Ukuhlaziywa nge-RPLC ehlanganiswe ne-electrospray ionization mass spectrometry, i-RC-LH1 complex yalungiswa nge-Rps ye-wild-type. Ngokusebenzisa indlela egadangiswe ngaphambilini (16), ukugxila kwamaphrotheyini akhiqizwa kumaseli we-palustris bekuyi-2 mg ml-1 ku-20 mM Hepes (pH 7.8), i-100 mM NaCl kanye ne-0.03% (w/v) β- (ukuhlaziywa kwe-Bio-Rad) ) DDM. Ngokuya kwephrothokholi yomkhiqizi, sebenzisa i-2D purification kit (GE Healthcare, USA) ukukhipha i-10 μg yamaphrotheyini ngendlela yokutjhisa, bese uncibilikisa i-precipitate ku-20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile kanye nama-20% (v/v) wamanzi. Ama-microlitha amahlanu ahlaziywa nge-RPLC (Dionex RSLC) ehlanganiswe ne-mass spectrometry (Maxis UHR-TOF, Bruker). Sebenzisa ikholomu ye-MabPac 1.2×100 mm (Thermo Fisher Scientific, UK) ukuhlukanisa ku-60°C kanye ne-100μlmin -1, nge-gradient ye-85% (v / v) isisombululo A [0.1% (v / v) FA kanye ne-0.02% (V/v) TFA isisombululo samanzi] ukuya ku-solvent (v / v) [0.1%(v/v) FA kanye no-0.02%(v/v) ku-90%(v/v) acetonitrile TFA] Ukusebenzisa umthombo ovamileko we-electrospray ionization kanye namapharamitha we-default isikhathi esingaphezu kwemizuzu ema-60, i-mass spectrometer ithola i-100 ukuya ku-2750 m/z (mass ratio-charge). Ngosizo lwe-ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), yenza imephu ye-mass spectrum kuma-subunits we-complex.
Amaseli akhuliswa ama-awa ama-72 ngaphasi kokukhanya okungu-100 ml NF-phasi (10μMm-2 s-1), okuphakathi (30μMm-2 s-1) nofana okuphezulu (300μMm-2 s-1). I-M22 medium (i-M22 medium lapho i-ammonium sulfate ingafakwa khona begodu i-sodium succinate ithathelwa indawo yi-sodium acetate) ebhodleleni eliyi-100 ml (23). Emijikelezweni emihlanu yama-30-s, ubuhlalu beglasi obuyi-0.1 micron bebufakwe ubuhlalu ngesilinganiso sevolumu engu-1:1 ukuhlukanisa amaseli begodu bapholiswa eqhweni imizuzu emi-5. Indaba engancibilikiko, amaseli angaphukileko kanye nama-glass beads kwasuswa ngokusebenzisa i-centrifugation ku-16,000 RCF imizuzu eli-10 ku-benchtop microcentrifuge. I-membrane yahlukaniswa nge-Ti 70.1 rotor ene-100,000 RCF ku-20 mM tris-HCl (pH 8.0) nge-40/15% (w/w) ye-sucrose gradient ama-awa ali-10.
Njengombana kuchaziwe emsebenzini wethu odlulileko, ukutholakala kwamasotja womzimba wethegi ye-His ku-PufW (16). Ngamafuphi, i-core complex ehlanjululweko (11.8 nM) nofana ulwelwesi oluqukethe ukugxila okufanako kwe-RC (okunqunywa yi-oxidation ukususa ukuhlukahluka okunciphileko nokumadanisa umthwalo ku-gel enebala) ku-2x SDS loading buffer (Merck, UK) ehlanjululwe kabili. Amaphrotheyini ahlukaniswe nge-replica 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). Ijeli yafakwa ibala nge-Coomassie Brilliant Blue (Bio-Rad, UK) ukulayisha nokubona i-RC-L subunit. Iphrotheyini esejeli yesibili yadluliselwa ku-methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) ukobana i-immunoassay. I-PVDF membrane yavalelwa ku-50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 kanye ne-5% (w / v) ye-skim milk powder, bese yafukanywa nge-anti-His primary antibody (ku-Dilute the antibody buffer [50 mM-HCl 7.6), 150 mM NaCl kanye ne-0.05% (v/v) Tween-20] ku-1:1000 A190-114A, Bethyl Laboratories, USA) ama-awa ama-4. Ngemva kokugeza amahlandla ama-3 imizuzu emi-5 nge-antibody buffer, i-membrane yahlanganiswa ne-horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (ehlanjululwe nge-1:10,000 ku-antibody buffer) Ukufukamela ukuvumela ukutholakala (imizuzu emi-5 ngemva kokugeza ka-3 ku-antibody buffer) kusetjenziswa i-WAR EST 2000. i-chemiluminescence substrate (Cyanagen, Italy) kanye ne-Amersham Imager 600 (GE Healthcare, UK).
Ngokudweba ukusabalala kwamandla we-gel ngayinye enebala nofana umzila we-immunoassay, ukuhlanganisa indawo engaphasi kwe-peak nokubala isilinganiso sokuqina kwe-RC-L (ijeli enebala) kanye ne-Protein-W (immunoassay), ku-ImageJ (57) Isebentisa isithombe. Ama-ratio la atjhugululwa aba ma-molar ratio ngokuthatha bona i-ratio ye-RC-L ukuya kuphrotheyini-W kusampula ye-RC-LH114-W emsulwa beyiyi-1:1 begodu yenza isethi yedatha yoke ibe yinto ejayelekileko.
Ukuthola izinto ezingezelelweko ze-atikili le, sibawa uqale i-http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
Le yi-atikili evulekileko esabalaliswa ngaphasi kwemibandela ye-Creative Commons Attribution License. I-atikili ivumela ukusetjenziswa okungakavunyelwa, ukusabalaliswa, nokukhiqizwa kabutjha kiyo nanyana ngiyiphi indlela ngaphasi kombandela wokobana umsebenzi wekuthomeni uvezwe kuhle.
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Umbuzo lo usetjenziselwa ukuhlola bona usivakatjhi begodu uvimbele ukuthunyelwa kwe-spam okuzenzakalelayo.
U-David JK Swainsbury, u-Park Qian, u-Philip J. Jackson, u-Kaitlyn M. Faries, u-Dariuzz M. Niedzwiedzki, u-Elizabeth C. Martin, u-David A. Farmer, u-Lorna A. Malone, u-Rebecca F. Thompson, u-Neil A. Ranson, u-Daniel P. Canniffe, u-Mark J. Dickman, u-Dewey Holten, u-Christine Kirma, u-Christine, u-Chritch, u-Andrew Hunter.
Isakhiwo esiphezulu sesithiyo sokukhanya 1 esiyinkimbinkimbi esikhungweni sokuphendula sinikela ukuzwisisa okutjha ngamandla we-quinone.
U-David JK Swainsbury, u-Park Qian, u-Philip J. Jackson, u-Kaitlyn M. Faries, u-Dariuzz M. Niedzwiedzki, u-Elizabeth C. Martin, u-David A. Farmer, u-Lorna A. Malone, u-Rebecca F. Thompson, u-Neil A. Ranson, u-Daniel P. Canniffe, u-Mark J. Dickman, u-Dewey Holten, u-Christine Kirma, u-Christine, u-Chritch, u-Andrew Hunter.
Isakhiwo esiphezulu sesithiyo sokukhanya 1 esiyinkimbinkimbi esikhungweni sokuphendula sinikela ukuzwisisa okutjha ngamandla we-quinone.
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