Ikheli lamanje: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Isikhungo sokusabela esihlanganisa ukukhanya 1 (RC-LH1) siyingxenye eyinhloko ye-photosynthetic yamagciwane e-purple phototrophic. Sethule izakhiwo ezimbili ze-cryo-electron microscopy ze-RC-LH1 complex ezivela ku-Rhodopseudomonas palustris. Isakhiwo sesisombululo se-2.65-Å se-RC-LH114-W complex siqukethe izihibe ze-subunit LH1 eziyi-14 ezizungeze i-RC, eziphazanyiswa yi-protein W, kuyilapho i-complex engena-protein-W ingukwakheka kwe-RC ngokuphelele izungezwe yi-RC. I-loop ye-subunit LH1 eyi-16 evaliwe. Ukuqhathaniswa kwalezi zakhiwo kunikeza ukuqonda nge-dynamics ye-quinone ku-RC-LH1 complex, kufaka phakathi izinguquko zokwakheka ezazingacaciswanga ngaphambilini lapho kuhlanganiswa i-quinone endaweni ye-RC QB, kanye nendawo yezindawo zokubopha ze-quinone ezisizayo, ezisiza ukuzidlulisela ku-RC. Isakhiwo esiyingqayizivele se-protein ye-W sivimbela ukuvalwa kwe-loop ye-LH1, ngaleyo ndlela sidale isiteshi sokusheshisa ukushintshaniswa kwe-quinone/quinolone.
Amandla anikezwa yi-photosynthesis angasekela cishe konke ukuphila emhlabeni, futhi anamandla amakhulu e-biotechnology yelanga. Ngenkathi ekhuthaza i-photosynthesis yomhlaba wonke, amabhaktheriya e-purple phototrophic nawo abonisa izindlela ezahlukene zamandla kanye namakhono okuguqula umzimba. Angagwema i-photosynthesis futhi akhule njengoba amabhaktheriya e-heterotrophic ebumnyameni, angalungisa i-nitrogen ne-carbon dioxide, akhiqize i-hydrogen, futhi onakalise ama-compounds e-aromatic (1-3). Ukuze kunikezwe amandla kulezi zinqubo, ukukhanya kumele kuguqulwe ngokushesha nangendlela efanele kube amandla amakhemikhali. Le nqubo iqala lapho i-antenna complex ebamba ukukhanya imunca ukukhanya futhi idlulisela amandla abanjwe esikhungweni sokusabela (RC), ngaleyo ndlela iqala ukuhlukaniswa kweshaja (4 - 7). Iyunithi eyisisekelo ye-photosynthesis kuma-bacteria e-purple phototrophic yakhiwe uhlobo lwe-RC 2, oluzungezwe i-light-harvesting complex 1 (LH1), eyakha i-RC-LH1 core complex. I-LH1 yakhiwa uhla lwe-αβ heterodimers ezigobile, ngayinye ebopha i-chlorophyll (BChl) ama-molecule amabili kanye ne-carotenoid eyodwa noma ezimbili (8-12). I-antenna elula ye-LH1 iqukethe ama-heterodimer angu-16 noma angu-17 e-αβ azungeze i-RC (9-13) ku-loop evaliwe, kodwa kwezinye izinhlanganisela eziyinhloko, ama-peptide e-transmembrane aphazamisa ukuqhubeka kwe-LH1 ezungezile, ngaleyo ndlela akhuthaza ukusabalala kwe-quinol/quinone phakathi kwe-RC kanye ne-cytochrome bc1 complex (11, 13-15). Isitshalo se-phototrophic esinsomi i-Rhodopseudomonas (Rps.) siyisibonelo sezinto eziphilayo esingaqonda amandla kanye nokudluliswa kwama-electron okusekela i-photosynthesis. Isakhiwo sokuqala sekristalu se-Rps. Imodeli ye-palustris RC-LH1 complex yi-RC, ezungezwe yi-heterodimeric LH1 loops engu-15, ephazanyiswa yiprotheni engaziwa ebizwa ngokuthi “iProtheyini W” (14). IProtheyini-W kamuva yabonakala njenge-RPA4402, okuyiprotheyini engu-10.5kDa engaziwa ene-transmembrane helices ezintathu ezibikezelwe (TMH) (16). Siphakamisa ukuqamba kabusha i-gene rpa4402 efaka iphrotheni W ibe yi-pufW ukuze ihambisane ne-nomenclature esetshenziselwa ama-gene efaka i-RC-L, M (pufL, pufM) kanye ne-LH1α, β (pufA, pufB) subunits. Ngokuthakazelisayo, i-protein-W ikhona kuphela cishe ku-10% we-RC-LH1, okwembula ukuthi i-Rps. palustris ikhiqiza ama-complex amabili ahlukene e-RC-LH1. Lapha, sibika izakhiwo ze-cryo-EM (cryo-EM) ezine-resolution ephezulu zama-complex amabili ayinhloko, enye ene-protein W kanye nama-heterodimer angu-14 e-αβ, enye engenayo iphrotheni W kanye ne-closed 16 Heterodimer LH1 loop. Isakhiwo sethu simelela ushintsho oluncane ekuqondeni i-RC-LH1 complex ye-Rps. palustris, ngoba sihlaziye inani labantu elifanayo lohlobo ngalunye futhi sinesisombululo esanele sokwabela ngokucacile i-peptide ngayinye kanye nama-pigment aboshwe kanye nama-lipid nama-quinone ahlobene. Ukuqhathaniswa kwalezi zakhiwo kubonisa ukuthi amaprotheni amathathu e-TMH-W angatholakalanga kunoma iyiphi enye i-RC-LH1 complex kuze kube manje akhiqiza isiteshi se-quinone ukusheshisa ukushintshaniswa kwe-quinone/quinolone. Izindawo eziningana zokubopha i-lipid ne-quinone ezigciniwe zitholiwe, futhi sembule ushintsho olusha lokwakheka ngemva kokuhlanganiswa kwe-quinone ne-RC, okungase kufane ne-photosystem II (PSII) RC yezinto eziphilayo ezine-oxygenated phototrophic. Okutholakele kwethu kunikeza ukuqonda okusha nge-kinetics yokubopha kwe-quinone/quinolone kanye nokushintshaniswa ku-RC-LH1 core complex yamagciwane e-purple phototrophic.
Ukuze kube lula ukutadisha okuningiliziwe kwama-complex amabili atholakala ku-Rps. palustris, sihlukanisa i-RC-LH1 ngayinye ngezindlela ze-biochemical. I-protein W-deficient complex (ebizwa ngokuthi i-ΔpufW) yahlanzwa kusuka ku-strain engenalo i-pufW gene (16), futhi kungakhiqizwa i-RC-LH1 complex eyodwa kuphela. I-protein W-containing complex ikhiqizwa yi-strain. I-protein W yalolu hlobo iguqulwa nge-tag ye-His engu-10x ekugcineni kwayo kwe-C, ukuze i-protein W-containing complex ihlanganiswe ngempumelelo ne-protein W eningi entula ngokuvimba insimbi. I-complex ihlukaniswe ngempumelelo (16) Affinity Chromatography (IMAC).
Njengoba kuboniswe kuMfanekiso 1, zombili lezi zakhiwo ziqukethe i-RC encane ezintathu (RC-L, RC-M kanye ne-RC-H) ezizungezwe yi-antenna ye-LH1. Isakhiwo se-2.80-A sesakhiwo esingenayo i-protein-W sibonisa ama-heterodimer angu-16 e-αβ, akha i-loop evaliwe ye-LH1 ezungeze i-RC, kamuva okubizwa ngokuthi i-RC-LH116 complex. Isakhiwo se-2.65Å sesakhiwo esiqukethe i-protein-W sine-LH1 engu-14-heterodimer ephazanyiswe yi-protein-W, kamuva okubizwa ngokuthi i-RC-LH114-W.
(A no-B) Ukumelwa kobuso benhlanganisela. (C no-D) Ama-pigment ahlanganisiwe abonakala ezindongeni. (E no-F) Ama-complex abonwa ebusweni be-cytoplasmic anama-peptide nama-subunit e-LH1 amelwe kumakhathuni, futhi abhalwe ngezinombolo ngokwewashi kusukela ku-protein-W gap [okuhambisana ne-Rba numbering. sphaeroides complex (13)]. Ku-LH1-α, umbala we-subunit yeprotheni uphuzi; ku-LH1-β, umbala we-subunit yeprotheni uluhlaza okwesibhakabhaka; ku-protein-W, iphrotheni ibomvu; ku-RC-H, iyi-cyan; ku-RC-L, iyi-Orange; ku-RC-M, iyi-magenta. Ama-Cofactor amelelwa yizinduku, oluhlaza lumelela i-BChl kanye ne-BPh ama-molecule, onsomi umelela ama-carotenoid, kanti ophuzi umelela ama-molecule e-UQ10. (G no-H) Umbono okhulisiwe we-protein-W gap esifundeni esilinganayo se-RC-LH114-W complex (G) kanye ne-RC-LH116 complex (H). Ama-cofactor aboniswa ngesimo sokugcwaliswa kwesikhala, i-quinone e-chelated iboniswa ngombala oluhlaza okwesibhakabhaka. Igebe le-protein-W ligqanyiswa umugqa oluhlaza okwesibhakabhaka onamachashazi ku-(G), kanti izimbobo ezincane lapho i-quinone/quinolol isakazeka khona eringini ye-LH116 zigqanyiswa umugqa omnyama onamachashazi ku-(H).
Isithombe 1 (A no-B) sibonisa i-RC ezungezwe ama-array avulekile noma avaliwe ama-heterodimers e-LH1αβ, ngalinye lawo libopha ama-BChl amabili kanye ne-carotenoid eyodwa (Isithombe 1, C no-D). Izifundo zangaphambilini zibonise ukuthi i-Rps iyinhlanganisela ye-LH1. Endleleni ye-biosynthetic ye-spirulina xanthin, lezi zinhlobo ziqukethe izinhlobo ezixubile zama-carotenoid (17). Kodwa-ke, i-spiropyrroxanthin iyi-carotenoid eyinhloko futhi ubuningi bayo buyanelisa. Ngakho-ke, sikhethe ukwenza imodeli ye-spiroxanthin kuzo zonke izindawo zokubopha ze-LH1. Ama-polypeptide e-alpha ne-beta angama-TMH angawodwa anezindawo ezimfushane zangaphandle ze-membrane (Isithombe 1, A, B, E, no-F). Nakuba ubuningi bezinsalela ezingu-17 ku-C-terminus bungazange bubonwe, i-alpha polypeptide yahlukaniswa kusuka ku-Met1 kuya ku-Ala46 kuzo zombili izinhlanganisela. I-β polypeptide yehlisiwe kusuka ku-Gly4 kuya ku-Tyr52 ku-RC-LH116, kanye nokusuka ku-Ser5 kuya ku-Tyr52 ku-RC-LH114-W. Akukho ukuminyana kwe-3 noma 4 N-terminal noma i-13 C-terminal residues ebonwe (Isithombe S1). Ukuhlaziywa kwe-mass spectrometry ye-complex exubile ye-RC-LH1 elungiselelwe kusuka kuhlobo lwe-wild-type kubonise ukuthi indawo engekho yayiwumphumela wokuqhekeka kwe-heterologous kwala ma-peptide (Isithombe S1 kanye ne-S2). I-N-terminal formlation ye-α-Met1 nayo yabonwa (f). Ukuhlaziywa kubonise ukuthi i-α-peptide iqukethe ama-residues fMet1 kuya ku-Asp42/Ala46/Ala47/Ala50, kanti i-β-peptide iqukethe ama-residues Ser2 kuya ku-Ala53, okuvumelana kahle nemephu yobuningi be-EM yokushisa okuphansi.
Ukuhlanganiswa kwe-α-His29 kanye ne-β-His36 kwenza ama-BChl abhekane ubuso nobuso; i-αβ heterodimer ngayinye ihlangana nomakhelwane bayo ukuze yakhe i-loop evulekile (RC-LH114-W) noma i-loop evaliwe (RC-LH116) ezungeze i-RC. I-exciton coupled pigment array (Isithombe 1, C kanye no-D). Uma kuqhathaniswa ne-877 nm band ye-RC-LH114-W, i-880 nm absorption red shift ye-RC-LH116 ingu-3 nm (Isithombe 2A). Kodwa-ke, i-circular dichroism spectrum icishe ifane (Isithombe 2B), okubonisa ukuthi nakuba kukhona umehluko ocacile phakathi kwama-loop avulekile navaliwe, indawo yasendaweni yama-BChl ifana kakhulu. I-absorption redshift ingaba umphumela wokunyakaza okuncishisiwe kokushisa kanye nokuqina okwandisiwe ku-loop evaliwe (18, 19), ushintsho ekuhlanganisweni kwe-pigment okubangelwa yi-loop evaliwe (20, 21), noma inhlanganisela yale miphumela emibili (11).
(A) I-Ultraviolet/ebonakalayo/eseduze kwe-infrared absorption spectrum, ama-peak awo aphawulwe ngama-pigment awo ahambisanayo futhi ajwayelekile esicongweni se-BPh ku-775 nm. (B) I-circular dichroism spectrum ijwayelekile esicongweni se-BChl absorbance ku-805 nm. (C no-D) Ama-spectra e-ΔA akhethiwe kusukela kuma-spectra okumunca axazululiwe ngesikhathi se-RC-LH114-W complex (C) kanye ne-RC-LH116 complex (D). Ukuze kuqhathaniswe kangcono, wonke ama-spectra ajwayelekile ku-∆A we-−A ku-0.2 ps. (E) Izinga le-cytochrome c2 oxidation ngemva kokushiswa kwemisebe phambi kwamazinga ahlukahlukene e-UQ2 (bheka Umfanekiso S8 ukuthola idatha eluhlaza). (F) Kumaseli akhuliswe ngaphansi kokukhanya okuphansi, okuphakathi noma okuphezulu (10, 30 noma 300μMm-2 s-1, ngokulandelana), ama-subunit e-protein W kanye ne-RC-L ku-complex ehlanziwe kanye nesilinganiso se-membrane esihlukanisiwe. Nquma izinga leprotheyini nge-SDS-polyacrylamide gel electrophoresis kanye ne-immunoassay (bheka uMfanekiso S9 ukuthola idatha eluhlaza). Nquma isilinganiso esihlobene ne-RC-LH114-W complex ehlanziwe. Isilinganiso se-stoichiometric se-RC-L ku-protein-W ye-complex singu-1:1.
Ama-BChls asendaweni 1 ku-loop ye-αβ14 eguquliwe ye-RC-LH114-W (Isithombe 1, A, C, kanye no-E) asondele kakhulu ku-RC primary donor (P) ngo-6.8Å kune-BChls efanayo ku-RC-LH116 (Isithombe 1, B, D, kanye no-F, kanye nesithombe S3); noma kunjalo, i-kinetics yokumuncwa kwesikhashana yalezi zakhiwo ezimbili ikhombisa ukuthi ku-RC-LH114-W kanye ne-RC-LH116, ama-constants wesikhathi sokudlulisa amandla okuvusa amandla kusuka ku-LH1 kuya ku-RC angama-40 ±4 kanye no-44±3 ps (Isithombe 2). , C kanye no-D, Isithombe S4 kanye neThebula S2). Akukho mehluko obalulekile ekudlulisweni kwe-elekthronikhi ngaphakathi kwe-RC (Isithombe S5 kanye nombhalo owengeziwe ohlobene). Sisola ukuthi ukuhambisana okuseduze kwesikhathi sokudlulisa amandla phakathi kwe-LH1 kanye ne-RC-P kungenxa yebanga elifanayo, i-engeli kanye namandla angase abe khona eningi le-BChl kuma-loop amabili e-LH1. Kubonakala sengathi ukuhlola iphethini yamandla e-LH1 ukuze ufinyelele ibanga elincane akusheshi kunokudluliswa kwamandla okuqondile kusuka ezindaweni ezingafanele kuya ku-RC. I-loop ye-LH1 evulekile ku-RC-LH114-W ingase futhi idlule ekunyakazeni kokushisa okuncane ngaphansi kwezimo zokushisa eziphansi ukuze kuhlaziywe isakhiwo, futhi kukhona ukwakheka kwendandatho ye-αβ14 ende ekushiseni kwegumbi kusukela ebangeni lokushintsha umbala lama-βBChls endaweni ye-RC 1.
I-RC-LH116 complex iqukethe ama-BChl angu-32 kanye nama-carotenoid angu-16, futhi ukuhlelwa kwayo konke kufana nalokho okutholwe ku-Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 strain (PDB ID 7C9R) (12) kanye ne-green algae (Blc.viridis) (PDB ID 6ET5) (10). Ngemva kokuqondanisa, kwabonakala ukuphambuka okuncane kuphela ezikhundleni zama-αβ heterodimers, ikakhulukazi u-1-5, 15, no-16 (Isithombe S6). Ukuba khona kwe-protein-W kunomthelela omkhulu esakhiweni se-LH1. Ama-TMH ayo amathathu axhunywe yizihibe ezimfushane, i-N-terminal ohlangothini lwe-lumen lwe-complex kanye ne-C-terminal ohlangothini lwe-cytoplasmic (Izithombe 1A no-3, A kuya ku-D). Iphrotheni-W ikakhulukazi ayithandi amanzi (Isithombe 3B), kanti i-TMH2 kanye ne-TMH3 zixhumana ne-LH1αβ-14 ukuze zakhe ubuso be-transmembrane (Isithombe 3, B kanye no-E kuya ku-G). I-interface yakhiwe kakhulu yi-Phe, Leu kanye ne-Val residues esifundeni se-transmembrane. Lezi zinsalela zihlanganiswe ngama-amino acids angathandi amanzi kanye nama-pigment e-αβ-14. Ezinye izinsalela ze-polar nazo zinegalelo ekusebenzisaneni, okuhlanganisa isibopho se-hydrogen phakathi kwe-W-Thr68 kanye ne-β-Trp42 ebusweni be-cavity eyinkimbinkimbi (Isithombe 3, F kanye no-G). Ebusweni be-cytoplasm, i-Gln34 iseduze neqembu le-keto lama-carotenoid e-αβ-14. Ngaphezu kwalokho, i-molecule ye-n-dodecyl β-d-maltoside (β-DDM) yaxazululwa, futhi umsila wayo ongathandi amanzi wanwetshwa waya ku-interface phakathi kwe-protein-W kanye ne-αβ-14, kanti umsila we-lipid ungase utholakale emzimbeni. Siphinde saphawula ukuthi izindawo ze-C-terminal resolution ze-protein W kanye ne-RCH zisondelene kakhulu, kodwa azikho ngaphakathi kobubanzi bokwakha ukusebenzisana okuthile (Isithombe 1, A kanye no-E). Kodwa-ke, kungase kube nokusebenzisana kuma-amino acid e-C-terminal angaxazululwa ala maprotheni amabili, okungase kuhlinzeke ngendlela yokuqoqana kwe-protein-W ngesikhathi sokuhlanganiswa kwe-RC-LH114-W complex.
(A) I-Protein-W, ebhekene ne-interface ne-LH1αβ14 ngesimo sekhathuni, inochungechunge oluseceleni olufana nenduku (obomvu), oluboniswe engxenyeni yomdwebo we-electrostatic potential (ubuso obumpunga obusobala obunezinga le-contour elingu-0.13). (B) I-Protein-W emelelwe ubuso obunombala we-hydrophobic. Izindawo ezi-polar nezishajekile ziboniswa nge-cyan, izindawo ze-hydrophobic ziboniswa ngombala omhlophe, kanti izindawo ze-hydrophobic kakhulu ziboniswa ngombala ophuzi. (C no-D) I-Protein-W emelelwe kukhathuni, ukuma kwayo kufana nokuku-(A) (C), futhi ijikeleziswa ngo-180° (D). Ngokwesikhundla esikulolu chungechunge, izinsalela ezihlukanisayo zamukela uhlelo lombala we-rainbow, lapho i-N-terminal iluhlaza okwesibhakabhaka kanti i-C-terminal ibomvu. (E) I-Protein-W ngokubuka okufanayo nokuku-(A), kanti izinsalela esibonakalayo se-protein-W:LH1 zimelelwe yizinduku ezinezimpawu ezinamathiselwe. (F) I-Protein-W ijikeleziswa ngo-90° uma kuqhathaniswa no-(E) kanye no-LH1αβ14 emelelweni yekhathuni, futhi uma kuqhathaniswa nezinsalela zesixhumi esibonakalayo emelelweni yebha. Izinsalela ezilengayo ezivela ku-beta polypeptide ziphawulwe. I-cofactor iboniswa njengebha ehambisana nombala weSithombe 1, i-β-DDM ebolile iboniswa ngompunga, kanti umoya-mpilo uboniswa ngobomvu. (G) Umbono ku-(F) ujikeleziswa ngo-180°, kanye nezinsalela ezivelele ze-alpha polypeptide ephawulwe.
I-Protein-W ithatha indawo ye-αβ heterodimer (eyesi-15 ku-Figure 1F), ngaleyo ndlela ivimbele ukuvalwa kwe-loop nokutshekisa ama-heterodimer amathathu okuqala e-αβ. Kwabonwa ukuthi i-engeli yokuthambekela ephezulu ye-heterodimer yokuqala ye-αβ-1 uma kuqhathaniswa nefilimu evamile yayingu-25° kuya ku-29° (Figure 1, A kanye no-E), eyakhiwa ukuthambekela okungu-2° kuya ku-8° kwe-αβ-1 ku-RC A sharp contrast-LH116 (Figure 1, B kanye no-F). Ama-heterodimer esibili nawesithathu athambekele ku-12° kuya ku-22° kanye no-5° kuya ku-10°, ngokulandelana. Ngenxa yesithiyo se-RC esiqinile, ukuthambekela kwe-αβ-1 akubandakanyi i-pair yesibili ye-αβ (ehambisana ne-αβ ye-16 ku-Figure 1F), ngaleyo ndlela kwakha igebe elicacile endandatho ye-LH1 (Figure 1, A kanye no-E). Ngenxa yokuntuleka kwama-heterodimer amabili e-αβ, ahambisana nokulahlekelwa ama-BChl amane nama-carotenoid amabili, ayikho i-carotenoid ebopha ku-subunit ephikisiwe ye-αβ-1, okuholela kundandatho ye-LH114-W equkethe ama-carotenoid angu-13 i-Vegetarian nama-BChl angu-28. Izilinganiso zesinqumo sendawo zama-complexes amabili ezifundeni ze-αβ1 kuya ku-7 ziphansi kunezo ze-loop ye-LH1 esele, okungabonisa ukuqina kwe-subunit ye-LH1 eduze nendawo ye-RC QB (Isithombe 4).
Izithombe ze-RC-LH114-W (A no-B) kanye ne-RC-LH116 (C no-D) ziboniswa kusukela ekubukeni okufanayo okuphezulu/ohlangothini (A no-B) (A no-C) kanye nobuso bomgodi wesithombe 1. (B no-D). Izinkinobho ezinemibala ziboniswe ngakwesokudla.
Enye i-complex eyinhloko kuphela enesilinganiso se-stoichiometric esingu-1:14 yi-Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Kodwa-ke, iphrotheni i-W ne-PufX azinawo ubudlelwano obucacile, futhi zinomthelela omkhulu ezakhiweni zazo ze-LH1. I-PufX iyi-TMH eyodwa enesizinda se-cytoplasmic se-N-terminal esisebenzisana nohlangothi lwe-cytoplasmic lwe-RC-H subunit (13) endaweni ehambisana ne-Rps. palustris LH116αβ-16. I-PufX idala isiteshi sokushintshana kwe-quinone/quinolone phakathi kwe-RC-LH1 kanye ne-cytochrome bcl complex futhi ikhona kuyo yonke i-Rba. sphaeroides core complex (13). Nakuba i-interface ye-monomer-monomer iku-Rba. I-sphaeroides RC-LH1-PufX dimer itholakala endaweni yokubopha yeprotheyini W ku-RC-LH114-W, kanti igebe elibangelwa yiPufX kanye neprotheyini-W lisendaweni efanayo (Isithombe S7A). Igebe ku-RC-LH114-W liphinde lihambisane nesiteshi se-quinone esicatshangelwayo (8) sePseudomonas rosea LH1, esakhiwe ngama-peptide angahlobene neprotheyini W noma iPufX (Isithombe S7B). Ngaphezu kwalokho, isiteshi se-quinone ku-Blc. I-LH1 eluhlaza e-emerald eyakhiwe ngokukhipha i-γ subunit eyodwa (7) itholakala endaweni efanayo (Isithombe S7C). Nakuba iqondiswa amaprotheni ahlukene, ukubonakala kwalezi ziteshi ze-quinone/quinolol endaweni efanayo ku-RC-LH1 complex kubonakala kuyisibonelo sokuvela okuguquguqukayo, okubonisa ukuthi igebe elidalwe yiprotheyini W lingasebenza njengesiteshi se-quinone.
Igebe eliku-loop ye-LH114-W livumela ukwakheka kwesifunda se-membrane esiqhubekayo phakathi kwesikhala sangaphakathi se-RC-LH114-W complex kanye ne-membrane enkulu (Isithombe 1G), kunokuxhumanisa izizinda ezimbili nge-pore yamaprotheni njengakuma-protein. I-RC-LH116 complex ifana ne-Tch. Needle-like complex evaliwe (22) (Isithombe 1H). Njengoba ukusabalala kwe-quinone nge-membrane kushesha kunokusabalala ngesiteshi esincane samaprotheni, i-loop evulekile ye-LH114-W ingavumela ukujika kwe-RC okusheshayo kune-loop evaliwe ye-LH116, futhi ukusabalala kwe-quinone ku-RC kungase kuvinjelwe kakhulu. Ukuze sihlole ukuthi iphrotheni W iyathinta yini ukuguqulwa kwama-quinone nge-RC, senze i-cytochrome oxidation assay ekugxilweni okuthile kwe-ubiquinone 2 (UQ2) (i-analogue ye-UQ10 yemvelo enomsila omfushane we-isoprene) (Isithombe 2E). Nakuba ukuba khona kwe-quinone e-chelated kuvimbela ukunqunywa okunembile kwe-Michaelis constant ebonakalayo (i-RC-LH114-W kanye ne-RC-LH116 zifanelekela i-0.2±0.1μM kanye ne-0.5±0.2μM, ngokulandelana), izinga eliphezulu le-RC-LH114-W (4.6±0.2 e-RC-1 s-1) likhulu ngo-28±5% kune-RC-LH116 (3.6±0.1 e-RC-1 s-1).
Ekuqaleni silinganisele ukuthi i-protein-W ikhona cishe ku-10% we-core complex (16); lapha, amazinga okuhlala kwamaseli okukhula anokukhanya okuphansi, okuphakathi, kanye nokukhanya okuphezulu angu-15±0.6%, 11±1% kanye no-0.9±0.5, ngokulandelana % (Isithombe 2F). Ukuqhathaniswa kwenani le-mass spectrometry kubonise ukuthi ukungezwa kwethegi ye-histidine akuzange kunciphise ubuningi be-protein-W uma kuqhathaniswa nezinhlobo ze-wild-type (P = 0.59), ngakho-ke la mazinga awayona into yokwenziwa kwe-protein-W eguquliwe (Isithombe S10). Kodwa-ke, lokhu kuhlala okuphansi kwe-protein-W ku-RC-LH1 complex kungavumela amanye ama-RC ukuthi aguquke ngesivinini esisheshayo, ngaleyo ndlela kuncishiswe ukushintshaniswa kwe-quinone/quinolone okuhamba kancane ku-RC-LH116 complex. Siqaphele ukuthi izinga lokuhlala kokukhanya okuphezulu alihambisani nedatha yakamuva ye-transcriptomics, ekhombisa ukuthi ukubonakaliswa kwezakhi zofuzo ze-pufW kuyanda ngaphansi kokukhanya okunamandla (Isithombe S11) (23). Umehluko phakathi kokubhalwa kwe-pufW kanye nokufakwa kwe-protein-W ku-RC-LH1 complex kuyadida futhi kungabonisa ukulawulwa okuyinkimbinkimbi kwe-protein.
Ku-RC-LH114-W, ama-cardiolipin angu-6 (CDL), ama-phosphatidylcholine angu-7 (POPC), i-phosphatidylglycerol engu-1 (POPG) kanye nama-molecule angu-29 e-β-DDM abekwa futhi aklanywe kuwo ama-CDL angu-6, ama-POPC angu-24, ama-POPG angu-2 kanye nama-βDDM angu-12. I-RC-LH116 (Isithombe 5, A kanye no-B). Kulezi zakhiwo ezimbili, i-CDL icishe itholakale ohlangothini lwe-cytoplasmic lwe-complex, kuyilapho i-POPC, i-POPG kanye ne-β-DDM zitholakala kakhulu ohlangothini lwe-luminal. Ama-molecule amabili e-lipid kanye ne-detergent ahlukaniswe esifundeni se-αβ-1 kuya ku-αβ-6 se-RC-LH114-W complex (Isithombe 5A), kanti amahlanu ahlukaniswe esifundeni esilinganayo se-RC-LH116 (Isithombe 5B). Kutholakale ama-lipid engeziwe ngakolunye uhlangothi lwe-complex, ikakhulukazi i-CDL, aqongelelwe phakathi kwe-RC ne-αβ-7 kuya ku-αβ-13 (Isithombe 5, A kanye no-B). Amanye ama-lipids axazululwe ngokwesakhiwo kanye nezihlanzi zitholakala ngaphandle kwendandatho ye-LH1, futhi amaketanga e-acyl axazululwe kahle adlula phakathi kwama-subunit e-LH1, abizwa ngokuthi i-β-DDM ku-RC-LH114-W, futhi achazwa ngokuthi i-β-DDM ku-RC A ingxube ye-β-DDM kanye ne-POPC-LH116. Izikhundla ezifanayo zama-lipids axegayo kanye nezihlanzi esakhiweni sethu zibonisa ukuthi ziyizindawo zokubopha ezibalulekile ngokomzimba (Isithombe S12A). Izikhundla zama-molecule alinganayo ku-Tch nazo zinokuvumelana okuhle. Okumnene kanye ne-Trv. I-Strin 970 RC-LH1s (Isithombe S12, B kuya ku-E) (9, 12) kanye nezinsalela ze-hydrogen-bonding zeqembu lekhanda le-lipid zibonise ukulondolozwa okuhle kakhulu ekulinganisweni kokulandelana (Isithombe S13), okubonisa ukuthi i-Conserved CDL ebopha ku-RC (24), lezi zindawo zingagcinwa ku-RC-LH1 complex.
(A no-B) Ama-peptide e-RC-LH114-W (A) kanye ne-RC-LH116 (B) amelelwa ngamakhathuni, kanti imibala imelelwa yizinduku, kusetshenziswa uhlelo lombala oluku-Figure 1. Ama-lipid aboniswa ngombala obomvu, kanti ama-detergent aboniswa ngombala ompunga. I-UQ eboshelwe ezindaweni ze-RC QA kanye ne-QB iphuzi, kuyilapho i-UQ ehlukanisiwe iluhlaza okwesibhakabhaka. (C no-D) Imibono efanayo ne-(A) kanye ne-(B), lapho ama-lipid esusiwe khona. (E kuya ku-G) Umbono okhulisiwe we-Q1(E), Q2(F) kanye ne-Q3(G) ovela ku-RC-LH116, ngamaketanga aseceleni athintana. Izibopho ze-hydrogen ziboniswa njengemigqa emnyama enezinhlayiya.
Ku-RC-LH116, kokubili i-RC QA kanye ne-QB UQ, ezihlanganyela ekudlulisweni kwama-electron enqubweni yokuhlukanisa ishaja, ziyabola ezindaweni zazo zokubopha. Kodwa-ke, ku-RC-LH114-W, i-QB quinone ayikaxazululwa futhi kuzoxoxwa ngayo ngokuningiliziwe ngezansi. Ngaphezu kwama-QA kanye nama-QB quinone, ama-molecule amabili e-UQ axubile (atholakala phakathi kwama-RC kanye nama-LH1 rings) abekwe esakhiweni se-RC-LH114-W ngokweqembu lawo lamakhanda axazululiwe kahle (atholakala ku-Q1 kanye ne-Q2, ngokulandelana). Isithombe 5C). Amayunithi amabili e-isoprene abelwe ku-Q1, kanti imephu yobuningi ixazulula imisila ephelele ye-isoprene eyi-10 ye-Q2. Esakhiweni se-RC-LH16, ama-molecule amathathu e-UQ10 axubile (Q1 kuya ku-Q3, Isithombe 5D) axazululiwe, futhi wonke ama-molecule anobuningi obucacile kulo lonke umsila (Isithombe 5, D kuya ku-G). Kulezi zakhiwo ezimbili, izikhundla zamaqembu ekhanda le-quinone le-Q1 ne-Q2 zinokuvumelana okuhle kakhulu (Isithombe S12F), futhi zixhumana kuphela ne-RC. I-Q1 itholakala emnyango wegebe le-W le-RC-LH114-W (Isithombe 1G no-5, C, D no-E), kanti i-Q2 itholakala eduze nendawo yokubopha ye-QB (Isithombe 5, C, D) no-F). Izinsalela ze-L-Trp143 ne-L-Trp269 ezigciniwe ziseduze kakhulu ne-Q1 ne-Q2 futhi zinikeza ukusebenzisana okungenzeka kwe-π-stacking (Isithombe 5, E no-F, kanye nesithombe S12). I-L-Gln88, 3.0 Å kusuka ku-oxygen ekude ye-Q1, inikeza isibopho esinamandla se-hydrogen (Isithombe 5E); le nsalela igcinwe kuwo wonke ama-RC ngaphandle kobudlelwano obukude kakhulu (Isithombe S13). I-L-Ser91 ithathelwe indawo ngokulondolozayo i-Thr kwamanye ama-RC amaningi (Isithombe S13), ingu-3.8 Angstroms evela ku-methyl oxygen ye-Q1, futhi inganikeza izibopho ze-hydrogen ezibuthakathaka (Isithombe 5E). I-Q3 ibonakala ingenakho ukusebenzisana okuthile, kodwa itholakala esifundeni esingenamanzi phakathi kwe-RC-M subunit kanye ne-LH1-α subunit 5 kuya ku-6 (Isithombe 5, D kanye no-G). I-Q1, Q2 kanye ne-Q3 noma ama-quinone aseduze acwebezelayo nawo axazululiwe ku-Tch. Gentle, Trv. Strain 970 kanye ne-Blc. Isakhiwo se-iris (9, 10, 12) sikhomba indawo yokubopha ye-quinone esilondoloziwe ku-RC-LH1 complex (Isithombe S12G). Ama-UQ amahlanu aqhekekile ku-RC-LH116 avumelana kahle no-5.8±0.7 wenkimbinkimbi ngayinye enqunywe yi-high performance liquid chromatography (HPLC), kuyilapho ama-UQ amathathu aqhekekile ku-RC-LH114-W ephansi kune-. Inani elilinganisiwe lika-6.2±0.3 (Isithombe S14) libonisa ukuthi kukhona ama-molecule e-UQ angaxazululiwe esakhiweni.
Ama-polypeptide e-L kanye ne-M angama-pseudo-symmetric ngalinye liqukethe ama-TMH amahlanu futhi akha i-heterodimer ehlanganisa i-BChl dimer eyodwa, ama-monomers amabili e-BChl, ama-monomers amabili e-bacteriophage (BPh), kanye ne-non-Heme iron eyodwa kanye ne-UQ10 molecule eyodwa noma ezimbili. Ngenxa yokuba khona kwezibopho ze-hydrogen eqenjini le-terminal ketone kanye nokuqongelela kwalo okwaziwayo ku-Rps, ama-carotenoid afakwa ku-M-subunit, ebizwa ngokuthi i-cis-3,4-dehydroorhodopin. Izinhlobo (25). Isizinda se-membrane yangaphandle ye-RC-H sinamathele ku-membrane yi-TMH eyodwa. Isakhiwo se-RC sisonke sifana ne-subunit ezintathu ze-RC zezinhlobo ezihlobene (njenge-Rba). sphaeroides (PDB ID: 3I4D). Ama-macrocycle e-BChl kanye ne-BPh, umgogodla we-carotenoid kanye ne-non-heme iron kuyahlangana ngaphakathi kobubanzi bokuxazulula kwalezi zakhiwo, njengoba kwenza iqembu lekhanda le-UQ10 endaweni ye-QA kanye ne-QB quinone ku-RC-LH116 (Isithombe S15).
Ukutholakala kwezakhiwo ezimbili ze-RC ezinezinga elihlukile lokuhlala endaweni ye-QB kunikeza ithuba elisha lokuhlola izinguquko ezihambisanayo zokwakheka ezihambisana nokubopha kwe-QB quinone. Ku-RC-LH116 complex, i-QB quinone itholakala endaweni "eseduze" ngokuphelele (26), kodwa ukuhlukaniswa kwe-RC-LH114-W akunayo i-QB quinone. Ayikho i-QB quinone ku-RC-LH114-W, okumangazayo ngoba i-complex iyasebenza, ngaphezu kwe-RC-LH116 complex ene-QB quinone exazululwe ngokwesakhiwo. Nakuba izindandatho ezimbili ze-LH1 zihlangana cishe nama-quinone ayisithupha, amahlanu axazululwe ngokwesakhiwo endandatho ye-RC-LH116 evaliwe, kuyilapho amathathu kuphela enqunyelwe ngokwesakhiwo endandatho evulekile ye-RC-LH114-W. Lokhu kuphazamiseka kwesakhiwo okwandisiwe kungabonisa ukushintshwa okusheshayo kwezindawo ze-RC-LH114-W QB, i-quinone kinetics esheshayo eyinkimbinkimbi, kanye namathuba akhulayo okuwela i-LH1 loop. Siphakamisa ukuthi ukuntuleka kwe-UQ endaweni ye-RC QB ye-RC-LH114-W kungaba umphumela wenkimbinkimbi eyinkimbinkimbi futhi esebenzayo kakhulu, futhi indawo ye-QB ye-RC-LH114-W ivalwe ngokushesha ekuguqukeni kwe-UQ. Isigaba esithile (ukungena endaweni ye-QB kuvaliwe) sibonisa ukwakheka kwalo msebenzi.
Ngaphandle kwe-QB, ukujikeleza okuhambisanayo kwe-L-Phe217 endaweni engahambisani nokubopha kwe-UQ10, ngoba kuzobangela ukushayisana kwendawo neyunithi yokuqala ye-isoprene yomsila (Isithombe 6A). Ngaphezu kwalokho, izinguquko ezicacile zokwakheka okuyinhloko zisobala, ikakhulukazi i-helix de (i-helix emfushane ku-loop phakathi kwe-TMH D no-E) lapho i-L-Phe217 idluliselwa ephaketheni lokubopha le-QB kanye nokujikeleza kwe-L-Tyr223 (Isithombe 6A) Ukuze kuphulwe isibopho se-hydrogen nohlaka lwe-M-Asp45 futhi kuvaliwe umnyango wendawo yokubopha ye-QB (Isithombe 6B). I-Helix de pivots esisekelweni sayo, i-Cα ye-L-Ser209 ishintshwa ngo-0.33Å, kuyilapho i-L-Val221Cα ishintshwa ngo-3.52Å. Azikho izinguquko ezibonakalayo ku-TMH D no-E, ezingaba khona kuzo zombili izakhiwo (Isithombe 6A). Ngokwazi kwethu, lesi yisakhiwo sokuqala ku-RC yemvelo esivala indawo ye-QB. Ukuqhathaniswa nesakhiwo esiphelele (esiboshwe yi-QB) kukhombisa ukuthi ngaphambi kokuba i-quinone incishiswe, kudingeka ushintsho lokwakheka ukuze ikwazi ukungena ku-quinone. I-L-Phe217 iyajikeleza ukuze yakhe ukusebenzisana kwe-π-stacking neqembu lekhanda le-quinone, bese i-helix ishintshela ngaphandle, okuvumela uhlaka lwe-L-Gly222 kanye nochungechunge oluseceleni lwe-L-Tyr223 ukuthi bakhe inethiwekhi yesibopho se-hydrogen enesakhiwo sesibopho se-hydrogen esizinzile (Isithombe 6, A no-C).
(A) Ikhathuni ehambisanayo ye-hologram (uchungechunge lwe-L, uchungechunge lwe-orange/M, i-magenta) kanye nesakhiwo se-apo (grey), lapho izinsalela ezibalulekile ziboniswa khona ngesimo sokumelwa okufana nenduku. I-UQ10 imelelwe ibha ephuzi. Umugqa onamachashazi ukhombisa izibopho ze-hydrogen ezakhiwe kuso sonke isakhiwo. (B no-C) Ukumelwa kobuso be-apolipoprotein kanye nesakhiwo sendandatho yonke, kugqamisa i-oxygen yeketanga eliseceleni le-L-Phe217 ngombala oluhlaza okwesibhakabhaka kanye ne-L-Tyr223 ngombala obomvu, ngokulandelana. I-subunit ye-L i-orenji; ama-subunit e-M kanye ne-H awanombala. (D no-E) I-Apolipoprotein (D) kanye ne-RC QB ephelele (E) [umbala nge-(A) ngokulandelana] kanye ne-Thermophilus thermophilus PSII (eluhlaza okwesibhakabhaka, oluhlaza okwesibhakabhaka nge-quinone yepulasitiki; i-PDB ID: 3WU2) Qondanisa (58).
Ngokungalindelekile, nakuba izakhiwo eziningana zama-RC angenayo i-QB angenayo i-LH1 zitholakala, izinguquko zokwakheka ezibonwe kulolu cwaningo azikaze zibikwe ngaphambilini. Lokhu kufaka phakathi isakhiwo sokuncipha kwe-QB esivela ku-Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) kanye ne-Rba. sphaeroides (PDB ID: 1OGV) (29), zonke ezicishe zifane nesakhiwo sazo se-QB iyonke. Ukuhlolwa okuseduze kwe-3PRC kwembule ukuthi ama-molecule okuhlanzisa e-LDAO (Lauryl Dimethyl Amine Oxide) abopha emnyango wesikhundla se-QB, okungase kuvimbele ukuhlelwa kabusha kube ukwakheka okuvaliwe. Nakuba i-LDAO ingaboli endaweni efanayo ku-1EYS noma ku-1OGV, lawa ma-RC alungiswa kusetshenziswa insipho efanayo futhi ngenxa yalokho angaveza umphumela ofanayo. Isakhiwo sekristalu se-Rba. I-Sphaeroides RC ehlanganiswe ne-cytochrome c2 (PDB ID: 1L9B) nayo ibonakala inesayithi le-QB elivaliwe. Kodwa-ke, kulokhu, isifunda se-N-terminal se-RC-M polypeptide (esebenzisana nesayithi lokubopha le-QB ngesibopho se-H se-Tyr residue ku-Q helix) samukela isimo esingavamile, futhi ushintsho lwesimo se-QB aluhlolisiswa kabanzi (30). Okuqinisekisayo ukuthi asikaze silubone lolu hlobo lokuguqulwa kwe-M polypeptide esakhiweni se-RC-LH114-W, esicishe sifane nesifunda se-N-terminal se-RC-LH116 RC. Kufanele futhi kuqashelwe ukuthi ngemva kokususwa kwe-antenna ye-LH1 esekelwe ekuhlanzeni, ama-apolipoprotein RCs ku-PDB axazululwa, okwasusa amachibi angaphakathi e-quinone nama-lipids esikhaleni esiphakathi kwe-RC kanye nobuso bangaphakathi bendandatho ye-LH1 ezungezile (31, 32). I-RC ihlala isebenza ngoba igcina wonke ama-cofactor, ngaphandle kwe-quinone ye-QB ebolayo, engazinzile futhi evame ukulahleka ngesikhathi senqubo yokulungiselela (33). Ngaphezu kwalokho, kwaziwa ukuthi ukususwa kwe-LH1 kanye nama-lipid e-cyclic yemvelo ku-RC kungaba nomthelela emisebenzini, njengobude besikhathi obufushane be-P+QB-state ehlukaniswe ngeshaja (31, 34, 35). Ngakho-ke, sicabanga ukuthi ukuba khona kwendandatho ye-LH1 yendawo ezungeze i-RC kungagcina indawo ye-QB “evaliwe”, ngaleyo ndlela kugcinwe indawo yendawo eduze kwe-QB.
Nakuba i-apolipoprotein (ngaphandle kwe-QB quinone) kanye nesakhiwo esiphelele kumelela izithombe ezimbili kuphela zokuguquka kwesayithi le-QB, kunokuba kube uchungechunge lwezehlakalo, kunezinkomba zokuthi ukubopha kungavinjelwa ukuvimbela ukubopha kabusha yi-hydroquinone Ukuvimbela ukuvinjelwa kwe-substrate. Ukusebenzisana kwe-quinolol ne-quinone eduze kwesayithi le-QB le-apolipoprotein kungase kuhluke, okuholela ekwenqatshweni kwayo yi-RC. Sekuyisikhathi eside kuphakanyiswe ukuthi izinguquko zokwakheka zidlala indima ekubopheni nasekuncishisweni kwama-quinone. Amandla ama-RC aqandisiwe okunciphisa ama-quinone ngemva kokuphazamiseka kokuzivumelanisa nobumnyama (36); i-X-ray crystallography ikhombisa ukuthi lo monakalo ubangelwa ama-quinone e-QB abanjwe ekubumbeni "okukude" cishe ngo-4.5 Å kusukela endaweni esebenzayo ye-proximal (26), 37). Siphakamisa ukuthi lokhu kuhlanganiswa kokubopha okukude kuyisithombe sesimo esiphakathi phakathi kwe-apolipoprotein nesakhiwo sendandatho egcwele, okulandela ukusebenzisana kokuqala ne-quinone kanye nokuvulwa kwesayithi le-QB.
Uhlobo lwe-RC II olutholakala ku-PSII complex yamabhaktheriya athile e-phototrophic kanye ne-cyanobacteria, ulwelwe kanye nezitshalo lunokulondolozwa kwesakhiwo nokusebenzayo (38). Ukuqondana kwesakhiwo okuboniswe ku-Figure 6 (D no-E) kugcizelela ukufana phakathi kwama-RSII e-PSII kanye nesayithi le-QB le-bacterial RC complex. Lokhu kuqhathaniswa sekuyisikhathi eside kuyimodeli yokufunda izinhlelo ezihlobene kakhulu zokubopha nokunciphisa i-quinone. Izincwadi zangaphambilini ziphakamise ukuthi izinguquko zokwakheka zihambisana nokunciphisa i-PSII yama-quinone (39, 40). Ngakho-ke, uma sicabangela ukulondolozwa kokuziphendukela kwemvelo kwe-RC, le ndlela yokubopha engakaze ibonwe ngaphambili ingase isebenze nakwisayithi le-QB le-PSII RC ezitshalweni ze-phototrophic ezine-oxygen.
I-Rps ΔpufW (ukususwa kwe-pufW okungafakwanga ilebula) kanye ne-PufW-His (i-C-terminal 10x His-tagged protein-W evezwe ezinhlotsheni zemvelo ze-pufW locus). I-palustris CGA009 ichazwe emsebenzini wethu wangaphambilini (16). Lezi zinhlobo kanye nomzali we-isogenic wild-type zitholwe efrijini ngokufaka inani elincane lamaseli ku-PYE (ngalinye i-5 g litre -1) (ligcinwe ku-LB ku--80 °C, liqukethe i-50% (w/v) glycerol) protein, i-yeast extract kanye ne-succinate) agar [1.5% (w/v)] plate. Ipuleti lafakwa ubusuku bonke ebumnyameni ekamelweni lokushisa ngaphansi kwezimo ze-anaerobic, bese likhanyiswa ngokukhanya okumhlophe (~50 μmolm-2 s-1) okunikezwe yi-OSRAM 116-W halogen bulbs (RS Components, UK) izinsuku ezi-3 kuya kwezi-5 kuze kube yilapho kuvela ikoloni elilodwa. Ikoloni elilodwa lasetshenziswa ukugoma i-10 ml ye-M22+ medium (41) engezwe yi-0.1% (w/v) casamino acids (ezobizwa ngokuthi i-M22). Isilimo sakhuliswa ngaphansi kwezimo eziphansi ze-oxygen ebumnyameni ku-34°C ngokunyakaziswa ku-180 rpm amahora angu-48, kwathi i-70 ml yesilimo yagoma ngaphansi kwezimo ezifanayo amahora angu-24. Isilimo esithambile esinevolumu engu-1 ml sisetshenziselwa ukugoma i-30 ml ye-M22 medium ebhodleleni lengilazi elicwebezelayo elingu-30 ml eliyi-universal screw-top futhi sashiswa ngemisebe (~50μmolm-2 s-1) amahora angu-48 ngamandla angenamandla angcolile i-Stirring rod. Ngemuva kwalokho i-30 ml yesilimo yagoma nge-1 litre yesitshalo ngaphansi kwezimo ezifanayo, eyabe isisetshenziselwa ukugoma cishe amalitha angu-9 esilimo akhanyiswe ku-~200 μmolm-2 s-1 amahora angu-72. Amaseli avunwa nge-centrifugation ku-7132 RCF imizuzu engama-30, aphinde axhunywe ku-~10 ml we-20 mM tris-HCl (pH 8.0), futhi agcinwa ku--20°C kuze kube yilapho kudingeka.
Ngemva kokuncibilika, engeza amakristalu athile e-deoxyribonuclease I (Merck, UK), i-lysozyme (Merck, UK) kanye namaphilisi amabili e-Roche holoenzyme protease inhibitor (Merck, UK) kumaseli aphinde axhunywa. Kuseli lokucindezela laseFrance elingu-20,000 psi (Aminco, USA), amaseli aphazamiseka izikhathi ezingu-8 kuya kwezingu-12. Ngemva kokususa amaseli angaphukile kanye nemfucumfucu enganyibiliki ngokufaka i-centrifugation ku-18,500 RCF imizuzu eyi-15 ku-4°C, i-membrane yasuswa ku-lysate enombala ngokufaka i-centrifugation ku-113,000 RCF amahora ama-2 ku-43,000°C. Lahla ingxenye encibilikayo bese ufaka i-membrane enemibala ku-100 kuya ku-200 ml ye-20 mM tris-HCl (pH 8.0) bese uhlanganisa kuze kube yilapho kungekho ma-aggregates abonakalayo. I-membrane elengayo ifakwe ku-20 mM tris-HCl (pH 8.0) (Anatrace, USA) equkethe i-2% (w/v) β-DDM ihora eli-1 ebumnyameni ku-4°C ngokuxuba ngobumnene. Bese ufaka i-centrifuge ku-70°C ukuze uncibilikise i-150,000 RCF ku-4°C ihora eli-1 ukuze ususe okusele okungancibiliki.
I-membrane encibilikisayo evela ku-ΔpufW strain isetshenziswe kukholomu yokushintshana kwama-ion e-DEAE Sepharose angu-50 ml enevolumu ezintathu zamakholomu (CV) ye-binding buffer [20 mM tris-HCl (pH 8.0) equkethe u-0.03% (w / v) β-DDM]. Geza ikholomu ngama-buffer amabili okubopha e-CV, bese ugeza ikholomu ngama-buffer amabili okubopha aqukethe u-50 mM NaCl. I-RC-LH116 complex ikhishwe nge-gradient eqondile engu-150 kuya ku-300 mM NaCl (ku-binding buffer) ku-1.75 CV, kanti i-binding complex esele ikhishwe nge-binding buffer equkethe u-300 mM NaCl ku-0.5 CV. Qoqa i-spectrum yokumunca ephakathi kuka-250 no-1000 nm, gcina ingxenye enesilinganiso sokumunca (A880/A280) esikhulu kune-1 ku-880 kuya ku-280 nm, uyinciphise kabili ku-binding buffer, bese usebenzisa inqubo efanayo futhi kukholomu ye-DEAE On purification. Nciphisa izingxenye ngezilinganiso ze-A880/A280 eziphakeme kunezilinganiso ze-1.7 kanye ne-A880/A805 eziphakeme kune-3.0, yenza umjikelezo wesithathu wokushintshaniswa kwe-ion, bese ugcina izingxenye ngezilinganiso ze-A880/A280 eziphakeme kunezilinganiso ze-2.2 kanye ne-A880/A805 eziphakeme kune-5.0. I-complex ehlanjululwe kancane yagxishwa ku-~2 ml kusihlungi se-centrifugal se-Amicon 100,000 molecular weight cut-off (MWCO) (Merck, UK), yabe isilayishwa kukholomu yokungafaki usayizi we-Superdex 200 16/600 (GE Healthcare, US) equkethe i-200 mM NaCl buffer, yabe isikhishwa ku-buffer efanayo ku-1.5 CV. Qoqa ama-spectra okumunca engxenyeni yokungafaki usayizi, bese ugxilisa ama-spectra okumunca ngezilinganiso ze-A880/A280 ngaphezu kwezilinganiso ze-2.4 kanye ne-A880/A805 ngaphezu kwezilinganiso ze-5.8 kuya ku-100 A880, bese uwasebenzisa ngokushesha ukulungiselela noma ukugcina igridi ye-cryo-TEM Gcina ku--80°C kuze kube yilapho kudingeka.
I-membrane encibilikisayo evela ku-PufW-His strain isetshenziswe kukholomu ye-HisPrep FF Ni-NTA Sepharose engu-20 ml (20 mM tris-HCl (pH 8.0) equkethe i-200 mM NaCl kanye ne-0.03% (w/w)) ku-IMAC buffer (GE Healthcare). v) β-DDM]. Ikholomu yagezwa ngama-CV amahlanu e-IMAC buffer, kwabe sekusetshenziswa ama-CV amahlanu e-IMAC buffer aqukethe i-10 mM histidine. I-core complex yasuswa kukholomu ngama-buffer amahlanu e-IMAC aqukethe i-100 mM histidine. Ingxenye equkethe i-RC-LH114-W complex igxiliswe ku-~10 ml ethangini elivuselelwe elihlonyiswe ngesihlungi se-Amicon 100,000 MWCO (Merck, UK), yancibilikiswa izikhathi ezingu-20 nge-binding buffer, yabe seyengezwa ku-25 ml Kukholomu ye-DEAE Sepharose, kusetshenziswa ama-CV amane aboshelwe ku-buffer kusengaphambili. Geza ikholomu ngama-buffer amane okubopha i-CV, bese ukhipha i-complex kuma-CV ayisishiyagalombili ku-gradient eqondile engu-0 kuya ku-100 mM NaCl (ku-binding buffer), kanye nama-CV amane asele aqukethe i-100 mM binding buffer. Ama-complexes asele akhishwe ku-sodium chloride ehlanganiswe nesilinganiso se-A880/A280 esingaphezu kuka-2.4 kanye nesilinganiso se-A880/A805 esingaphezu kwama-fraction angu-4.6 agxilwe ku-~2 ml kusihlungi se-centrifugal se-Amicon 100,000 MWCO, futhi agcwaliswe nge-1.5 CV IMAC kusengaphambili. Ikholomu yokukhipha usayizi we-Buffer equilibrated Superdex 200 16/600, bese esuswa ku-buffer efanayo phezu kwe-1.5 CV. Qoqa ama-spectra okumunca ama-fractions angafakwanga ngobukhulu bese ugxilisa ama-spectra okumunca ngezilinganiso ze-A880/A280 ezingaphezu kuka-2.1 kanye nezilinganiso ze-A880/A805 ezingaphezu kuka-4.6 kuya ku-100 A880, asetshenziswa ngokushesha ekulungiseleleni igridi ye-TEM eqandisiwe noma agcinwe ku--80°C kuze kube yilapho kudingeka.
Ifriji yokucwilisa ye-Leica EM GP yasetshenziswa ukulungiselela amagridi e-TEM anokushisa okuphansi. I-complex yancibilikiswa ku-IMAC buffer ku-A880 yama-50, kwabe sekufakwa u-5μl ku-mesh yethusi esanda kukhishwa i-QUANTIFOIL 1.2/1.3 enekhabhoni (i-Agar Scientific, UK). Faka igridi ku-20°C kanye nomswakama ongu-60% imizuzwana engu-30, bese uyisula imizuzwana emi-3, bese uyicima ku-ethane ewuketshezi ku--176°C.
Idatha ye-RC-LH114-W complex iqoshwe ku-eBIC (Electronic Bioimaging Center) (British Diamond Light Source) nge-microscope ye-Titan Krios, esebenza nge-voltage esheshisa engu-300kV, enokukhulisa okulinganiselwe okungu-130,000× kanye namandla e- Khetha igebe elingu-20 eV. I-Gatan 968 GIF Quantum ene-K2 peak detector isetshenziswe ukuqopha izithombe kwimodi yokubala ukuqoqa idatha. Usayizi wephikseli olinganisiwe ungu-1.048Å, kanti izinga lomthamo lingu-3.83 e-Å-2s-1. Iqoqe i-movie ngemizuzwana eyi-11 futhi yahlukanisa yaba izingxenye ezingu-40. Sebenzisa indawo embozwe nge-carbon ukuze uphinde ugxilise i-microscope, bese uqoqa ama-movie amathathu ngembobo ngayinye. Sekukonke, kuqoqwe ama-movie angu-3130, anamanani e-defocus aphakathi kuka--1 no--3μm.
Idatha ye-RC-LH116 complex iqoqwe kusetshenziswa i-microscope efanayo e-Asterbury Biostructure Laboratory (eNyuvesi yaseLeeds, e-UK). Idatha iqoqwe ngendlela yokubala ngokukhulisa okungu-130 k, kanti usayizi wamaphikseli ulinganiswe ku-1.065 Å ngomthamo ongu-4.6 e-Å-2s-1. Ifilimu iqoshwe ngemizuzwana eyi-12 futhi yahlukaniswa yaba izingxenye ezingu-48. Sekukonke, amafilimu angu-3359 aqoqwe, anamanani e-defocus aphakathi kuka--1 no--3μm.
Konke ukucutshungulwa kwedatha kwenziwa kupayipi le-Relion 3.0 (42). Sebenzisa i-Motioncorr 2 (43) ukulungisa ukunyakaza kwe-beam ngokulinganisa idosi, bese usebenzisa i-CTFFIND 4.1 (44) ukuthola ipharamitha ye-CTF (umsebenzi wokudlulisa umehluko). Ama-photomicrograph ajwayelekile ngemva kwalezi zigaba zokuqala zokucubungula aboniswe ku-Figure 2. S16. Ithempulethi yokukhetha okuzenzakalelayo ikhiqizwa ngokukhetha ngesandla cishe amaphikseli angu-250 ezinhlayiya ezingu-1000 kufreyimu yamaphikseli angu-250 futhi akukho ukuhlukaniswa okubhekiselwe kukho okunezinhlangothi ezimbili (2D), ngaleyo ndlela kwenqatshwa lezo zigaba ezihlangabezana nokungcola kwesampula noma ezingenazo izici ezibonakalayo. Ngemuva kwalokho, ukukhethwa okuzenzakalelayo kwenziwa kuzo zonke izithombe ezincane, kanti i-RC-LH114-W yayiyizinhlayiya ezingu-849,359, kanti i-RC-LH116 complex yayiyizinhlayiya ezingu-476,547. Zonke izinhlayiya ezikhethiwe ziye zadlula emizuliswaneni emibili yokuhlukaniswa kwe-2D okungeyona ireferensi, futhi ngemva kokugijima ngakunye, izinhlayiya ezihlangabezana nendawo yekhabhoni, ukungcola kwesampula, okungekho zici ezicacile noma izinhlayiya ezihambisanayo kakhulu ziyalahlwa, okuholela ezinhlayiya ezingu-772,033 (90.9%) kanye no-359,678 (75.5%) zisetshenziselwa ukuhlukaniswa kwe-RC-LH114-W kanye ne-RC-LH116 nge-3D ngokulandelana. Imodeli yokuqala yokubhekisela ye-3D yakhiqizwa kusetshenziswa indlela ye-stochastic gradient desccent. Usebenzisa imodeli yokuqala njengereferensi, izinhlayiya ezikhethiwe zihlukaniswa ngezigaba ezine ku-3D. Usebenzisa imodeli kulesi sigaba njengereferensi, yenza ukuhluzwa kwe-3D ezinhlayiyeni esigabeni esikhulu kunazo zonke, bese usebenzisa isihlungi sokuqala se-15Å low-pass ukumboza indawo ye-solvent, engeza amaphikseli angu-6 emiphetho ethambile, bese ucubungula amaphikseli ngemuva kokulungisa umsebenzi wokudlulisa i-Gatan K2 peak Modulation we-top detector. Kusethi yedatha ye-RC-LH114-W, le modeli yokuqala ishintshwe ngokususa ubuningi obuqinile emaphethelweni emaski (engaxhunywanga ku-density ye-core complex ku-UCSF Chimera). Amamodeli aphumelelayo (izinqumo ze-RC-LH114-W kanye ne-RC-LH116 ziyi-3.91 kanye ne-4.16 Å, ngokulandelana) asetshenziswa njengereferensi yomzuliswano wesibili wokuhlukaniswa kwe-3D. Izinhlayiya ezisetshenzisiwe ziqoqwe ekilasini lokuqala le-3D futhi aziqukethe ubudlelwano obuqinile nomakhelwane. Ziyahambisana noma azinazo izici zesakhiwo ezisobala. Ngemuva komzuliswano wesibili wokuhlukaniswa kwe-3D, isigaba esinesinqumo esiphezulu sikhethiwe [Ku-RC-LH114-W, isigaba esisodwa siyizinhlayiya ezingu-377,703 (44.5%), ku-RC-LH116, kunezigaba ezimbili, eziphelele ziyizinhlayiya ezingu-260,752 (54.7%), Lapho zifana khona kuphela uma zihambisana ngemva kokujikeleza kokuqala ngomehluko omncane]. Izinhlayiya ezikhethiwe ziphinde zikhishwe ebhokisini lamaphikseli angu-400 bese zihlungwa ngokuhlungwa kwe-3D. Imaski ye-solvent ikhiqizwa kusetshenziswa isihlungi sokuqala se-15Å low-pass, ukunwetshwa kwemephu yamaphikseli angu-3 kanye nemaski ethambile yamaphikseli angu-3. Kusetshenziswa ukulungiswa kwe-CTF ngezinhlayiya ngayinye, ukulungiswa kokunyakaza kwezinhlayiya ngayinye kanye nomjikelezo wesibili wokulungiswa kwe-CTF ngezinhlayiya ngayinye, ukulungiswa kwe-3D, ukufihla i-solvent kanye nokucubungula ngemuva kwenziwa ngemuva kwesinyathelo ngasinye ukuze kuthuthukiswe ukuthungwa okubangelwayo. Kusetshenziswa inani elinqunyiwe le-FSC (Fourier Shell Correlation Coefficient) elingu-0.143, izinqumo zamamodeli okugcina e-RC-LH114-W kanye ne-RC-LH116 ziyi-2.65 kanye ne-2.80Å, ngokulandelana. Ijika le-FSC lemodeli yokugcina liboniswe kuMfanekiso 2. S17.
Zonke izilandelana zamaprotheni 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); Protein-W (PufW; UniProt ID: Q6N1K3). I-SWISS-MODEL (45) yasetshenziswa ukwakha imodeli ye-homology ye-RC, equkethe izilandelana zamaprotheni ze-RC-L, RC-M kanye ne-RC-H kanye nesakhiwo sekristalu se-Rba. I-sphaeroides RC yasetshenziswa njengethempulethi (PDB ID: 5LSE) (46). Sebenzisa ithuluzi elithi “fit map” ku-UCSF Chimera ukuze uvumelane nemodeli ekhiqizwe kumephu (47), uthuthukise isakhiwo seprotheyini, bese usebenzisa i-cofactor [4×BChl a (igama le-monomer library residue = BCL), 2×BPh a (BPH), uhlobo olulodwa noma ezimbili ze-UQ10 (U10), insimbi eyodwa engeyona i-heme (Fe) kanye nenye engu-3,4-dihydrohexacarbonylcholine (QAK)] sebenzisa i-Coot (48) ukuze wengeze. Njengoba i-QAK ingatholakali kulabhulali ye-monomer, yahlelwa ngepharamitha kusetshenziswa ithuluzi le-eLBOW ku-PHENIX (49).
Okulandelayo, kwakhiwa i-subunit ye-LH1. Ekuqaleni, ithuluzi lokwakha elizenzakalelayo ku-PHENIX (49) lasetshenziswa ukwakha ngokuzenzakalelayo ingxenye yochungechunge lwe-LH1 kusetshenziswa imephu kanye nochungechunge lwe-protein lwe-LH1-α kanye ne-LH1-β njengokufaka. Khetha i-subunit ye-LH1 ephelele kakhulu, uyikhiphe bese uyilayisha ku-Coot, wengeze ngesandla uhlu olungekho kulo, bese ulungisa isakhiwo sonke ngesandla ngaphambi kokwengeza ama-BCls amabili (BCL) kanye ne-spirilloxanthin (CRT) [ngokusho kwe-Rps The density ye-LH1 complex kanye nokuqukethwe kwe-carotenoid okwaziwayo. Izinhlobo (17)]. Kopisha i-subunit ephelele ye-LH1, bese usebenzisa i-UCSF Chimera “Docking Map Tool” ukuze udoke endaweni engeyona imodeli eseduze ye-LH1 density, bese uyilungisa ku-Coot; phinda inqubo kuze kube yilapho wonke ama-subunit e-LH1 esemodeliwe. Ngesakhiwo se-RC-LH114-W, ngokukhipha ubuningi obungafakwanga ku-Coot, iphrotheni ihlukaniswa kusuka ezingxenyeni ezisele ezingezona amaprotheni kumephu ye-USCF Chimera bese ithuluzi le-Autobuild lisetshenziselwa ukusungula imodeli yokuqala, kanye nama-subunit asele (protein-W) Modeling. Ku-PHENIX (49). Engeza noma yikuphi ukulandelana okungekho kumodeli ephumayo ku-Coot (48), bese ulungisa ngesandla yonke i-subunit. Ubuningi obungafakwanga obusele bufanelana nenhlanganisela yama-lipids (ID yomtapo wezincwadi we-PDB monomer we-CDL = CDL, POPC = 6PL kanye ne-POPG = PGT), i-detergent ye-β-DDM (LMT) kanye nama-molecule e-UQ10 (U10). Sebenzisa i-PHENIX optimization (49) kanye ne-manual optimization ku-Coot (48) ukuze uphelele imodeli yokuqala ephelele kuze kube yilapho izibalo zemodeli kanye nekhwalithi ebonakalayo yokulingana kungenakuthuthukiswa. Okokugcina, sebenzisa i-LocScale (50) ukuze ulole imephu yendawo, bese wenza eminye imijikelezo eminingana yokumodela ubuningi obungafakwanga kanye nokwenza ngcono okuzenzakalelayo kanye nokwesandla.
Ama-peptide, ama-cofactor kanye namanye ama-lipid nama-quinone abekwe ngaphakathi kobuningi bawo aboniswe kuZithombe 1 no-2. S18 kuya ku-S23. Ulwazi lwezibalo lwemodeli yokugcina luboniswe kuThebula S1.
Ngaphandle kokuthi kuchazwe ngenye indlela, ama-spectra okumunca i-UV/Vis/NIR aqoqwe ku-Cary60 spectrophotometer (e-Agilent, e-USA) ngezikhawu ze-1 nm kusukela ku-250 nm kuya ku-1000 nm kanye nesikhathi sokuhlanganiswa samasekhondi angu-0.1.
Nciphisa isampula ku-cuvette ye-quartz enendlela engu-2 mm eya ku-A880 ye-1, bese uqoqa i-spectrum yokumunca ephakathi kuka-400 no-1000 nm. Ama-spectra ayindilinga e-dichroic aqoqwe ku-Jasco 810 spectropolarimeter (eJasco, eJapan) ngezikhawu ze-1 nm phakathi kuka-400 nm no-950 nm ngesilinganiso sokuskena esingu-20 nm min-1.
I-molar extinction coefficient inqunywa ngokunciphisa i-core complex ibe yi-A880 engaba ngu-50. Nciphisa ivolumu engu-10μl ku-990μl binding buffer noma i-methanol, bese uqoqa i-absorption spectrum ngokushesha ukuze unciphise ukubola kwe-BChl. Okuqukethwe kwe-BChl kwesampula ngayinye ye-methanol kubalwe yi-extinction coefficient ku-771 nm ka-54.8 mM-1 cm-1, kwathi i-extinction coefficient yanqunywa (51). Hlukanisa i-BChl concentration elinganisiwe ngo-32 (RC-LH114-W) noma u-36 (RC-LH116) ukuze kunqunywe i-core complex concentration, esetshenziswa ukunquma i-absorption spectrum yesampula efanayo eqoqwe ku-buffer Extinction coefficient. parallel. Kuthathwe izilinganiso ezintathu eziphindaphindwayo zesampula ngayinye, kwathi i-absorption emaphakathi ye-BChl Qy maximum yasetshenziswa ekubalweni. I-coefficient yokuphela kwe-RC-LH114-W elinganiswe ku-878 nm ingu-3280±140 mM-1 cm-1, kanti i-coefficient yokuphela kwe-RC-LH116 elinganiswe ku-880 nm ingu-3800±30 mM-1 cm-1.
I-UQ10 yalinganiswa ngokwendlela eku-(52). Ngamafuphi, i-reverse phase HPLC (RP-HPLC) yenziwe kusetshenziswa uhlelo lwe-Agilent 1200 HPLC. Ncibilikisa cishe i-0.02 nmol ye-RC-LH116 noma i-RC-LH114-W ku-50μl ye-50:50 methanol:chloroform equkethe i-0.02% (w/v) ferric chloride, bese ufaka i-pre-equilibrated Beckman Coulter Ultrasphere ODS 4.6 mm Ncibilikisa ku-1 ml-1 min-1 ku-40°C ku-HPLC solvent (80:20 methanol:2-propanol) kukholomu engu-×25 cm. Yenza i-isocratic elution ku-HPLC solvent ukuze uqaphe ukumuncwa ku-275 nm (UQ10), 450 nm (carotenoids) kanye ne-780 nm (BChl) ihora eli-1. Isilinganiso esiphezulu ku-chromatogram engu-275 nm ngemizuzu engu-25.5 sahlanganiswa, esasingenazo ezinye izinto ezitholakalayo. Indawo ehlanganisiwe isetshenziselwa ukubala inani le-molar le-UQ10 elikhishwe ngokubhekisela ku-calibration curve ebalwe kusukela ekufakweni kwezindinganiso ezihlanzekile kusukela ku-0 kuya ku-5.8 nmol (Isithombe S14). Isampula ngayinye yahlaziywa ngamakhophi amathathu, futhi iphutha elibikiwe lihambisana ne-SD yesilinganiso.
Isixazululo esiqukethe i-RC-LH1 complex enomthamo omkhulu we-Qy wokumuncwa okungu-0.1 salungiswa nge-30 μM reduced horse heart cytochrome c2 (Merck, UK) kanye ne-0 kuya ku-50 μMUQ2 (Merck, UK). Amasampula amathathu angu-1-ml alungiswa ku-UQ2 concentration ngayinye futhi afakwa ubusuku bonke ebumnyameni ku-4°C ukuqinisekisa ukuzivumelanisa okuphelele nobumnyama ngaphambi kokulinganiswa. Isixazululo safakwa ku-OLIS RSM1000 modular spectrophotometer efakwe i-300 nm flame/500 line grating, i-1.24 mm inlet, i-0.12 mm middle kanye ne-0.6 mm outlet slits. Isihlungi se-pass eside esingu-600 nm sibekwa emnyango we-phototube yesampula kanye ne-reference photomultiplier tube ukuze kukhishwe ukukhanya okuvusa inkanuko. Ukumuncwa kwaqashwa ku-550 nm ngesikhathi sokuhlanganiswa esingu-0.15 seconds. Ukukhanya okuvusa inkanuko kukhishwa ku-880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) ngekhebula le-fiber optic ngamandla angu-90% ngesilawuli se-DC2200 (Thorlabs Ltd., UK) futhi kukhishwa emthonjeni wokukhanya nge-engeli engu-90° of. Ugongolo lokulinganisa luphambene nesibuko ukuze lubuyisele noma yikuphi ukukhanya okungamuncwanga yisampula ekuqaleni. Qapha ukumuncwa imizuzwana eyi-10 ngaphambi kokukhanya kwemizuzwana engama-50. Ngemuva kwalokho ukumuncwa kwaqashelwa imizuzwana engama-60 ebumnyameni ukuze kuhlolwe izinga i-quinolol elinciphisa ngalo i-cytochrome c23 + ngokuzenzakalelayo (bheka Umfanekiso S8 ukuthola idatha eluhlaza).
Idatha icutshungulwe ngokufaka isilinganiso sokuqala esiqondile ngaphakathi kwemizuzwana engu-0.5 kuya kwengu-10 (kuye ngokuthi i-UQ2 concentration) kanye nokulinganisa amazinga azo zonke amasampula amathathu ku-UQ2 concentration ngayinye. I-RC-LH1 concentration ebalwe yi-extinction coefficient efanele yasetshenziswa ukuguqula isilinganiso sibe ukusebenza kahle kwe-catalytic, okwadwetshwa ku-Origin Pro 2019 (OriginLab, USA), futhi kwafakwa kumodeli kaMichaelis-Menten ukuze kunqunywe amanani abonakalayo e-Km kanye ne-Kcat.
Ukuze kulinganiswe ukumuncwa kwesikhashana, isampula ye-RC-LH1 incibilikiswe ibe yi-~2μM ku-IMAC buffer equkethe i-sodium ascorbate engu-50 mM (Merck, USA) kanye ne-0.4 mM Terbutin (Merck, USA). I-Ascorbic acid isetshenziswa njengomnikeli wama-electron omhlatshelo, kanti i-tert-butaclofen isetshenziswa njenge-QB inhibitor ukuqinisekisa ukuthi umnikeli oyinhloko we-RC uhlala encishisiwe (okungukuthi, hhayi i-photooxidized) kuyo yonke inqubo yokulinganisa. Cishe i-3 ml yesampula ingezwa kuseli elijikelezayo elenziwe ngokwezifiso (cishe ububanzi obungu-0.1 m, i-350 RPM) enobude bendlela ye-optical engu-2 mm ukuqinisekisa ukuthi isampula endleleni ye-laser inesikhathi esanele sokuzivumelanisa nobumnyama phakathi kwama-pulse okuvusa inkanuko. Sebenzisa ama-pulse e-laser angu-~100-fs ukuze ukhulise uhlelo lwe-laser lwe-Ti: Sapphire (Spectra Physics, USA) ukuze uvuse isampula ku-880 nm ngesilinganiso sokuphindaphinda esingu-1 kHz (20 nJ ye-NIR noma i-100 nJ ye-Vis). Ngaphambi kokuqoqa idatha, veza isampula ekukhanyeni okuvusayo cishe imizuzu engama-30. Ukuvezwa kuzobangela ukungasebenzi kwe-QA (mhlawumbe kunciphisa i-QA kanye noma kabili). Kodwa sicela uqaphele ukuthi le nqubo ingashintsha ngoba ngemva kwesikhathi eside sokuzivumelanisa nobumnyama, i-RC izobuyela kancane emsebenzini we-QA. I-Helios spectrometer (Ultrafast Systems, USA) yasetshenziswa ukukala ama-transient spectra ngesikhathi sokulibaziseka esingu--10 kuya ku-7000 ps​​. Sebenzisa isofthiwe ye-Surface Xplorer (Ultrafast Systems, USA) ukuze uhlukanise amasethi edatha, bese uhlanganisa futhi ulinganise. Sebenzisa iphakheji yesofthiwe ye-CarpetView (Light Conversion Ltd., Lithuania) ukuze usebenzise isethi yedatha ehlanganisiwe ukuthola ama-spectra ahlukene ahlobene nokubola, noma sebenzisa umsebenzi ohlanganisa ama-exponents amaningi nempendulo yethuluzi ukuze ivumelane nokuguquka kwe-spectral ye-single-wavelength ku-Origin (OriginLab, USA).
Njengoba kushiwo ngenhla (53), kwalungiswa ifilimu ye-photosynthetic equkethe i-LH1 complex engenazo zombili i-RC kanye ne-antenna ye-LH2 engaphandle. I-membrane yancibilikiswa ku-20 mM tris (pH 8.0) yabe isifakwa ku-quartz cuvette enendlela ye-optical engu-2 mm. I-30nJ laser pulse yasetshenziswa ukuvusa isampula ku-540 nm ngesikhathi sokulibaziseka esingu--10 kuya ku-7000 ps. Cubungula isethi yedatha njengoba kuchaziwe ku-Rps. pal sample.
I-membrane yahlungwa nge-centrifugation ku-150,000 RCF amahora ama-2 ku-4°C, bese ukumuncwa kwayo ku-880 nm kwaphinde kwamiswa ku-20 mM tris-HCl (pH 8.0) kanye ne-200 mM NaCl. Ncibilikisa i-membrane ngokufaka kancane kancane ku-2% (w/v) β-DDM ihora eli-1 ebumnyameni ku-4°C. Isampula yahlungwa ku-100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) kuze kube yilapho kugcwele amaprotheni angu-2.5 mg ml-1 (ukuhlaziywa kwe-Bio-Rad). Ukucubungula okwengeziwe kwenziwa ngendlela eyanyatheliswa ngaphambilini (54), kwaqala ngokuhlungwa kwamaprotheni angu-50 μg abe yi-50 μl TEAB equkethe i-1% (w/v) sodium laurate (Merck, UK). Ngemva kokusebenzisa i-sonication imizuzwana engama-60, yancishiswa nge-5 mM tris(2-carboxyethyl)phosphine (Merck, UK) ku-37°C imizuzu engama-30. Nge-S-alkylation, faka isampula nge-10 mM methyl S-methylthiomethanesulfonate (Merck, UK) bese uyifaka kusuka kusisombululo sesitoko se-isopropanol esingu-200 mM imizuzu eyi-10 ekushiseni kwegumbi. Ukugaya kwe-Proteolytic kwenziwa ngokungeza ingxube ye-2 μg trypsin/endoproteinase Lys-C (Promega UK) futhi kwafakwa ku-37°C amahora ama-3. I-laurate surfactant yakhishwa ngokungeza i-50 μl ethyl acetate kanye ne-10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) kanye ne-vortexing imizuzwana engama-60. Ukuhlukaniswa kwesigaba kukhuthazwe nge-centrifugation ku-15,700 RCF imizuzu emi-5. Ngokwephrothokholi yomenzi, ikholomu ye-C18 spin (i-Thermo Fisher Scientific, UK) yasetshenziswa ukufutha ngokucophelela nokususa usawoti esigabeni esingezansi esiqukethe i-peptide. Ngemva kokomiswa nge-vacuum centrifugation, isampula yancibilikiswa ku-0.5% TFA kanye ne-3% acetonitrile, kwathi i-500 ng yahlaziywa yi-nanoflow RP chromatography ehlanganiswe ne-mass spectrometry kusetshenziswa amapharamitha esistimu achazwe ngaphambilini.
Sebenzisa i-MaxQuant v.1.5.3.30 (56) ukuze uthole ukuhlonza amaprotheni kanye nokulinganisa ukuze useshe isizindalwazi se-Rps. palustris proteome (www.uniprot.org/proteomes/UP000001426). Idatha ye-mass spectrometry proteomics ifakwe ku-ProteomeXchange Alliance ngokusebenzisa indawo yokugcina yabalingani be-PRIDE (http://proteomecentral.proteomexchange.org) ngaphansi kwesihlonzi sesethi yedatha i-PXD020402.
Ukuze kuhlaziywe yi-RPLC ehambisana ne-electrospray ionization mass spectrometry, i-RC-LH1 complex yalungiswa kusuka ku-wild-type Rps. Kusetshenziswa indlela eyanyatheliswa ngaphambilini (16), ukuhlushwa kwamaprotheni okukhiqizwe kumaseli e-palustris kwakuyi-2 mg ml-1 ku-20 mM Hepes (pH 7.8), i-100 mM NaCl kanye ne-0.03% (w/v) β- (Bio-Rad analysis)) DDM. Ngokwephrothokholi yomenzi, sebenzisa i-2D purification kit (GE Healthcare, USA) ukukhipha iphrotheni engu-10 μg ngendlela yokwehliswa, bese uncibilikisa i-precipitate ku-20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile kanye no-20% (v/v) wamanzi. Ama-microliter amahlanu ahlaziywe yi-RPLC (Dionex RSLC) ehlanganiswe ne-mass spectrometry (Maxis UHR-TOF, Bruker). Sebenzisa ikholomu ye-MabPac 1.2×100 mm (Thermo Fisher Scientific, UK) ukuze uhlukanise ku-60°C kanye ne-100μlmin -1, ene-gradient engu-85% (v / v) solvent A [0.1% (v / v) FA kanye ne-0.02% (V/v) TFA aqueous solution] kuya ku-85%(v/v) solvent B [0.1%(v/v) FA kanye ne-0.02%(v/v) ku-90%(v/v) acetonitrile TFA] Usebenzisa i-electrospray standard ionization source kanye namapharamitha azenzakalelayo isikhathi esingaphezu kwemizuzu engama-60, i-mass spectrometer ithola isilinganiso esingu-100 kuya ku-2750 m/z (mass-to-charge ratio). Ngosizo lwe-ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), hlela i-mass spectrum kuma-subunit e-complex.
Amaseli akhuliswe amahora angu-72 ngaphansi kokukhanya okungu-100 ml kwe-NF-low (10μMm-2 s-1), okuphakathi (30μMm-2 s-1) noma okuphezulu (300μMm-2 s-1). Okuphakathi kwe-M22 (okuphakathi kwe-M22 lapho i-ammonium sulfate ishiywa khona bese kuthi i-sodium succinate ithathelwe indawo yi-sodium acetate) ebhodleleni elingaphezulu kwesikulufo elingu-100 ml (23). Emijikelezweni emihlanu yamasekhondi angu-30, ubuhlalu bengilazi obuyi-0.1 micron bahlotshiswe ngobuhlalu ngesilinganiso sevolumu esingu-1:1 ukuze kuhlanzwe amaseli futhi kupholiswe eqhweni imizuzu emi-5. Izinto ezinganyibiliki, amaseli angaphukile kanye nobuhlalu bengilazi kwasuswa nge-centrifugation ku-16,000 RCF imizuzu eyi-10 ku-benchtop microcentrifuge. I-membrane yahlukaniswa ku-rotor ye-Ti 70.1 ene-RCF engu-100,000 ku-20 mM tris-HCl (pH 8.0) ene-gradient ye-sucrose engu-40/15% (w/w) amahora ayi-10.
Njengoba kuchaziwe emsebenzini wethu wangaphambilini, ukutholwa kwamasosha omzimba kwethegi likaHis ku-PufW (16). Ngamafuphi, i-complex ehlanzekile ye-core (11.8 nM) noma i-membrane equkethe ukuhlushwa okufanayo kwe-RC (okunqunywa yi-oxidation esusa i-spectrum yomehluko encishisiwe futhi ifanise umthwalo kujeli enemibala) ku-2x SDS loading buffer (Merck, UK) incishisiwe kabili. Amaprotheni ahlukaniswe kujeli ye-NuPage engu-12% bis-tris (Thermo Fisher Scientific, UK). Ijeli yagcotshwa nge-Coomassie Brilliant Blue (Bio-Rad, UK) ukuze ilayishe futhi ibone ngeso lengqondo i-RC-L subunit. Iphrotheni kujeli yesibili yadluliselwa ku-membrane ye-polyvinylidene fluoride (PVDF) eyenziwe nge-methanol (Thermo Fisher Scientific, UK) ukuze kuhlolwe i-immunoassay. I-membrane ye-PVDF ivinjiwe ku-50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 kanye no-5% (w / v) ubisi oluphuciwe, bese lufakwa nge-antibody eyinhloko ye-anti-His (ku-Dilute the antibody buffer [50 mM tris-HCl (pH 7.6), 150 mM NaCl kanye no-0.05% (v/v) Tween-20] ku-1:1000 A190-114A, Bethyl Laboratories, USA) amahora ama-4. Ngemva kokugeza izikhathi ezi-3 imizuzu emi-5 ku-antibody buffer, i-membrane yahlanganiswa ne-horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (exutshwe ne-1:10,000 ku-antibody buffer) Ukufukamela ukuze kutholakale (imizuzu emi-5 ngemva kokugeza oku-3 ku-antibody buffer) kusetshenziswa i-WESTAR ETA C 2.0 chemiluminescence substrate (Cyanagen, Italy) kanye ne-Amersham Imager 600 (GE Healthcare, UK).
Ngokudweba ukusatshalaliswa kokuqina kwejeli ngayinye enemibala noma umzila we-immunoassay, ukuhlanganisa indawo engaphansi kwesiqongo nokubala isilinganiso sokuqina kwe-RC-L (ijeli enemibala) kanye ne-Protein-W (immunoassay), ku-ImageJ (57) Cubungula isithombe. Lezi zilinganiso zaguqulwa zaba yizilinganiso ze-molar ngokucabanga ukuthi isilinganiso se-RC-L kuya ku-protein-W kusampula emsulwa ye-RC-LH114-W sasingu-1:1 futhi senza isethi yedatha yonke ibe ngokwejwayelekile ngokufanele.
Ukuze uthole izinto ezengeziwe zalesi sihloko, sicela ubheke ku-http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
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