Idilesi yangoku: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
I-reaction center light-harvesting complex 1 (RC-LH1) yeyona nto iphambili kwi-photosynthetic bacteria ye-purple phototrophic. Siqalise izakhiwo ezimbini ze-cryo-electron microscopy ze-RC-LH1 complex ezivela kwi-Rhodopseudomonas palustris. Isakhiwo sesisombululo se-2.65-Å se-RC-LH114-W complex siqulathe ii-subunit ezili-14 ze-LH1 loops ezijikeleze i-RC, eziphazanyiswa yi-protein W, ngelixa i-complex engena-protein-W iyinxalenye ye-RC ngokupheleleyo ejikelezwe yi-RC. I-loop ye-subunit ezili-16 ze-LH1 ivaliwe. Uthelekiso lwezi zakhiwo lubonelela ngengqiqo kwi-dynamics ye-quinone kwi-RC-LH1 complex, kubandakanya utshintsho olungazange luchazwe ngaphambili xa kuhlanganiswa i-quinone kwindawo ye-RC QB, kunye nendawo yeendawo zokubopha ze-quinone ezincedisayo, ezinceda ukuba zidluliselwe kwi-RC. Isakhiwo esahlukileyo se-protein ye-W sithintela ukuvalwa kwe-loop ye-LH1, ngaloo ndlela senza umjelo wokukhawulezisa utshintshiselwano lwe-quinone/quinolone.
Amandla anikezelwa yi-photosynthesis anokuxhasa phantse zonke izinto eziphilayo emhlabeni, kwaye anamandla amakhulu kwi-biotechnology yelanga. Ngelixa ikhuthaza i-photosynthesis yehlabathi, iibhaktheriya ezimfusa ze-phototrophic zikwabonisa iindlela ezahlukeneyo zamandla kunye nobuchule be-metabolic. Zingayiphepha i-photosynthesis kwaye zikhule njengebhaktheriya ezingafaniyo ebumnyameni, zinokulungisa i-nitrogen kunye ne-carbon dioxide, zivelise i-hydrogen, kwaye zonakalise iikhompawundi ze-aromatic (1-3). Ukuze kubonelelwe ngamandla kwezi nkqubo, ukukhanya kufuneka kuguqulwe ngokukhawuleza nangokufanelekileyo kube ngamandla eekhemikhali. Le nkqubo iqala xa i-antenna complex ebamba ukukhanya ifunxa ukukhanya kwaye idlulisela amandla abanjwe kwiziko lokusabela (RC), ngaloo ndlela iqala ukwahlukana kwetshaja (4 - 7). Iyunithi esisiseko ye-photosynthesis kwibhaktheriya ezimfusa ze-phototrophic yenziwe yi-type 2 RC, ejikelezwe yi-light-harvesting complex 1 (LH1), eyenza i-RC-LH1 core complex. I-LH1 yenziwe luluhlu lwee-heterodimers ezigobileyo ze-αβ, nganye kuzo ibopha i-chlorophyll ezimbini zebhaktheriya (BChl) iimolekyuli kunye ne-carotenoids enye okanye ezimbini (8-12). I-antenna elula ye-LH1 inee-heterodimer ezili-16 okanye ezili-17 ze-αβ ezijikeleze i-RC (9-13) kwi-loop evaliweyo, kodwa kwezinye ii-complexes eziphambili, ii-transmembrane peptides ziphazamisa ukuqhubeka kwe-LH1 ejikelezileyo, ngaloo ndlela zikhuthaza ukusasazeka kwe-quinol/quinone phakathi kwe-RC kunye ne-cytochrome bc1 complex (11, 13-15). Isityalo esinombala omfusa i-phototrophic i-Rhodopseudomonas (Rps.) sisidalwa esingumzekelo esinokuqonda amandla kunye nokudluliselwa kwe-electron okuxhasa i-photosynthesis. Isakhiwo sokuqala sekristale se-Rps. Imodeli ye-palustris RC-LH1 complex yi-RC, ejikelezwe zii-loops ezili-15 ze-heterodimeric LH1, eziphazanyiswa yiproteni engaziwayo ebizwa ngokuba yi-"Protein W" (14). I-Protein-W kamva yachongwa njenge-RPA4402, eyiproteni engachazwanga ye-10.5kDa ene-helices ezintathu ze-transmembrane eziqikelelweyo (TMH) (16). Sicebisa ukuba sitshintshe igama le-rpa4402 gene encoding protein W ibe yi-pufW ukuze ihambelane negama elisetyenziselwa ii-genes encoding RC-L, M (pufL, pufM) kunye ne-LH1α, β (pufA, pufB) subunits. Okubangela umdla kukuba, i-protein-W ikhona kuphela kwi-10% ye-RC-LH1, nto leyo ebonisa ukuba i-Rps. palustris ivelisa ii-complexes ezimbini ezahlukeneyo ze-RC-LH1. Apha, sixela izakhiwo ze-cryo-EM (cryo-EM) ezinesisombululo esiphezulu see-complexes ezimbini eziphambili, enye ine-protein W kunye ne-14 αβ heterodimers, enye ingenayo i-protein W kunye ne-closed 16 Heterodimer LH1 loop. Isakhiwo sethu simele utshintsho oluncinci ekuqondeni i-RC-LH1 complex ye-Rps. palustris, kuba sihlalutye inani labantu abalinganayo kwi-variant nganye kwaye sinesisombululo esaneleyo sokwabela ngokucacileyo i-peptide nganye kunye nee-bound pigments kunye ne-lipids kunye ne-quinones ezinxulumene nayo. Uthelekiso lwezi zakhiwo lubonisa ukuba iiproteni ezintathu ze-TMH-W ezingakhange zifumaneke nakweyiphi na enye i-RC-LH1 complex ukuza kuthi ga ngoku zivelisa itshaneli ye-quinone ukukhawulezisa utshintsho lwe-quinone/quinolone. Iindawo ezininzi zokubopha i-lipid kunye ne-quinone ezigciniweyo zichongiwe, kwaye sityhile utshintsho olutsha emva kokudibana kwe-quinone kunye ne-RC, enokufaneleka kwi-photosystem II (PSII) RC yezinto eziphilayo ezine-oxygenated phototrophic. Iziphumo zethu zibonelela ngolwazi olutsha malunga ne-kinetics yokubopha i-quinone/quinolone kunye nokutshintsha kwi-RC-LH1 core complex yebhaktheriya ye-purple phototrophic.
Ukuze kube lula uphononongo oluneenkcukacha lwee-complexes ezimbini ezifumaneka kwi-Rps. palustris, sahlula i-RC-LH1 nganye ngeendlela ze-biochemical. I-protein W-deficient complex (ebizwa ngokuba yi-ΔpufW apha) yahlanjululwa kwi-strain engenalo i-pufW gene (16), kwaye i-RC-LH1 complex enye kuphela enokwenziwa. I-protein W-containing complex iveliswa yi-strain. I-protein W yale strain iguqulwa nge-10x His tag kwi-C-terminus yayo, ukuze i-protein W-containing complex idityaniswe ngempumelelo ne-protein W eninzi engenalo ngokuyithintela isinyithi. I-complex yahlulwe ngempumelelo (16) Affinity Chromatography (IMAC).
Njengoko kubonisiwe kuMfanekiso 1, zombini ezi complexes ziqulathe i-RC encinci ezintathu (RC-L, RC-M kunye ne-RC-H) ezijikelezwe yi-antenna ye-LH1. Isakhiwo se-2.80-A se-complex engena-protein-W sibonisa ii-heterodimers ezili-16 ze-αβ, ezenza i-loop ye-LH1 evaliweyo ejikeleze i-RC, emva koko ebizwa ngokuba yi-RC-LH116 complex. Isakhiwo se-2.65Å se-complex equlethe i-protein-W sine-14-heterodimer LH1 ephazanyiswe yi-protein-W, emva koko ebizwa ngokuba yi-RC-LH114-W.
(A kunye no-B) Ukubonakaliswa komphezulu wekhompawundi. (C kunye no-D) Iipigment ezidityanisiweyo ezivezwa kwiintonga. (E kunye no-F) Iicomplex ezibonwa kumphezulu we-cytoplasmic zineepeptides kunye nee-subunits ze-LH1 ezimelelwe kwiikhathuni, kwaye zibalwa ngokwewotshi ukusuka kwi-protein-W gap [ehambelana ne-Rba numbering. sphaeroides complex (13)]. Kwi-LH1-α, umbala we-protein subunit umtyheli; kwi-LH1-β, umbala we-protein subunit uluhlaza okwesibhakabhaka; kwi-protein-W, iproteni ibomvu; kwi-RC-H, yi-cyan; kwi-RC-L, yi-Orange; kwi-RC-M, yi-magenta. Ii-Cofactors zimelelwe ziintonga, uhlaza lumela i-BChl kunye ne-BPh iimolekyuli, i-purple imela ii-carotenoids, kwaye i-yellow imela iimolekyuli ze-UQ10. (G kunye no-H) Umbono okhulisiweyo we-protein-W gap kummandla olinganayo we-RC-LH114-W complex (G) kunye ne-RC-LH116 complex (H). Ii-cofactors ziboniswa ngendlela yokuzalisa isithuba, i-quinone etyheli iboniswa ngombala oluhlaza okwesibhakabhaka. Umsantsa weproteni-W ugqanyiswa ngumgca oluhlaza okwesibhakabhaka onamachaphaza ku-(G), kwaye imingxunya emincinci apho i-quinone/quinolol isasazeka khona kwiringi ye-LH116 igqanyiswa ngumgca omnyama onamachaphaza ku-(H).
Umfanekiso 1 (A kunye no-B) ubonisa i-RC ejikelezwe zii-arrays ezivulekileyo okanye ezivaliweyo ze-LH1αβ heterodimers, nganye kuzo ibopha ii-BChl ezimbini kunye ne-carotenoid enye (Umfanekiso 1, C kunye no-D). Izifundo zangaphambili zibonise ukuba i-Rps yi-LH1 complex. Kwindlela ye-biosynthetic ye-spirulina xanthin, ezi ntlobo ziqulathe ii-carotenoids ezixutyiweyo (17). Nangona kunjalo, i-spiropyrroxanthin yi-carotenoid elawulayo kwaye uxinano lwayo luyanelisa. Ke ngoko, sikhethe ukwenza imodeli ye-spiroxanthin kuzo zonke iindawo zokubopha ze-LH1. Ii-polypeptides ze-alpha kunye ne-beta zii-TMHs ezingatshatanga ezineendawo ezimfutshane zangaphandle ze-membrane (Umfanekiso 1, A, B, E, kunye no-F). Nangona uxinano lweentsalela ezili-17 kwi-C-terminus lungakhange lubonwe, i-alpha polypeptide yahlulwa ukusuka kwi-Met1 ukuya kwi-Ala46 kuzo zombini ii-complexes. I-β polypeptide yehlisiwe ukusuka kwi-Gly4 ukuya kwi-Tyr52 kwi-RC-LH116, kwaye ukusuka kwi-Ser5 ukuya kwi-Tyr52 kwi-RC-LH114-W. Akukho xinano lwe-3 okanye i-4 N-terminal okanye i-13 C-terminal residues ebonwe (Umfanekiso S1). Uhlalutyo lwe-mass spectrometry lwe-mixed RC-LH1 complex elungiselelwe kwi-wild-type strain lubonise ukuba indawo elahlekileyo yayiyimiphumo yokuqhekeka kwe-heterologous kwezi peptides (Umfanekiso S1 kunye no-S2). I-N-terminal formylation ye-α-Met1 nayo yabonwa (f). Uhlalutyo lubonise ukuba i-α-peptide inee-residues fMet1 ukuya kwi-Asp42/Ala46/Ala47/Ala50, kwaye i-β-peptide inee-residues Ser2 ukuya kwi-Ala53, ezivumelana kakuhle nemephu ye-low-temperature EM density.
Ukudibanisa kwe-α-His29 kunye ne-β-His36 kwenza ii-BChs zijongane; i-αβ heterodimer nganye ihlangana nabamelwane bayo ukuze yenze i-open loop (RC-LH114-W) okanye i-closed loop (RC-LH116) ejikeleze i-RC. I-exciton coupled pigment array (Umfanekiso 1, C kunye no-D). Xa kuthelekiswa ne-877 nm band ye-RC-LH114-W, i-880 nm absorption red shift ye-RC-LH116 yi-3 nm (Umfanekiso 2A). Nangona kunjalo, i-circular dichroism spectrum iphantse ifane (Umfanekiso 2B), nto leyo ebonisa ukuba nangona kukho umahluko ocacileyo phakathi kwe-open loops ne-closed loops, imeko-bume yendawo ye-BChs iyafana kakhulu. I-absorption redshift inokuba sisiphumo sokunciphisa intshukumo yobushushu kunye nokuqina okwandisiweyo kwi-closed loop (18, 19), utshintsho kwi-pigment coupling olubangelwa yi-closed loop (20, 21), okanye indibaniselwano yezi ziphumo zimbini (11).
(A) I-Ultraviolet/ebonakalayo/ekufutshane ne-infrared absorption spectrum, apho iincopho zazo ziphawulwe ngeepigment zazo ezihambelanayo kwaye zilungelelaniswe kwi-BPh peak kwi-775 nm. (B) I-circular dichroism spectrum ilungelelaniswe kwi-BChl absorbance kwi-805 nm. (C kunye no-D) I-ΔA spectra ekhethiweyo kwi-time-resolved absorption spectra ye-RC-LH114-W complex (C) kunye ne-RC-LH116 complex (D). Ukuze kube nokuthelekiswa okungcono, zonke ii-spectra zilungelelaniswe kwi-∆A ye-−A kwi-0.2 ps. (E) Isantya se-cytochrome c2 oxidation emva kokukhanyiswa phambi koxinzelelo olwahlukeneyo lwe-UQ2 (jonga uMfanekiso S8 ukuze ufumane idatha eluhlaza). (F) Kwiiseli ezikhuliswe phantsi kokukhanya okuphantsi, okuphakathi okanye okuphezulu (10, 30 okanye 300μMm-2 s-1, ngokulandelanayo), ii-protein W kunye ne-RC-L subunits kwi-complex ehlanjululweyo kunye nomlinganiselo we-membrane owahlulwe. Misela inqanaba leproteni nge-SDS-polyacrylamide gel electrophoresis kunye ne-immunoassay (jonga uMfanekiso S9 ukuze ufumane idatha eluhlaza). Misela umlinganiselo ohambelana ne-RC-LH114-W complex ecociweyo. Umlinganiselo we-stoichiometric we-RC-L kwi-protein-W ye-complex yi-1:1.
Ii-BChls ezikwindawo yoku-1 kwi-deformed αβ14 loop ye-RC-LH114-W (Umfanekiso 1, A, C, kunye no-E) zisondele kwi-RC primary donor (P) nge-6.8Å kunee-BChls ezifanayo kwi-RC-LH116 (Umfanekiso 1, B, D, kunye no-F, kunye nomfanekiso S3); nangona kunjalo, i-transient absorption kinetics zezi complexes zimbini zibonisa ukuba kwi-RC-LH114-W kunye ne-RC-LH116, ii-excitation energy transfer time constants ukusuka kwi-LH1 ukuya kwi-RC zingama-40 ±4 kunye nama-44±3 ps (Umfanekiso 2). , C kunye no-D, Umfanekiso S4 kunye neTheyibhile S2). Akukho mahluko abalulekileyo ekudlulisweni kwe-elektroniki ngaphakathi kwe-RC (Umfanekiso S5 kunye nombhalo ongezelelweyo ohambelanayo). Sicinga ukuba ukuhambelana okusondeleyo kwexesha lokudluliselwa kwamandla phakathi kwe-LH1 kunye ne-RC-P kungenxa yomgama ofanayo, i-angle kunye namandla anokubakho uninzi lwe-BChl kwii-loops ezimbini ze-LH1. Kubonakala ngathi ukuhlola ipateni yamandla e-LH1 ukuze kufikelelwe kumgama omncinci akukhawulezi kunokudluliselwa kwamandla ngokuthe ngqo ukusuka kwiindawo ezingalungelanga ukuya kwi-RC. I-loop ye-LH1 evulekileyo kwi-RC-LH114-W isenokudlula kwintshukumo yobushushu engabalulekanga phantsi kweemeko zobushushu obuphantsi ukuze kuhlalutywe ulwakhiwo, kwaye kukho ukwakheka kwendandatho ye-αβ14 ende kubushushu begumbi ukusuka kumgama we-pigmentation we-βBChls kwindawo ye-RC 1.
I-RC-LH116 complex iqulethe ii-BChls ezingama-32 kunye nee-carotenoids ezili-16, kwaye ulungelelwaniso lwayo lulonke lufana nolo lufunyenwe kwi-Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 strain (PDB ID 7C9R) (12) kunye ne-green algae (Blc.viridis) (PDB ID 6ET5) (10). Emva kokulungelelaniswa, kuphela ukuphambuka okuncinci kwizikhundla ze-αβ heterodimers okubonwe, ngakumbi i-1-5, 15, kunye ne-16 (Umfanekiso S6). Ubukho be-protein-W bunempembelelo enkulu kwisakhiwo se-LH1. Ii-TMH zayo ezintathu ziqhagamshelwe zii-short loops, kunye ne-N-terminal kwicala le-lumen ye-complex kunye ne-C-terminal kwicala le-cytoplasmic (Imifanekiso 1A kunye ne-3, A ukuya ku-D). Iproteni-W ikakhulu ayithandi manzi (Umfanekiso 3B), kwaye i-TMH2 kunye ne-TMH3 zidibana ne-LH1αβ-14 ukuze zenze umphezulu we-transmembrane (Umfanekiso 3, B kunye no-E ukuya ku-G). Ujongano lwenziwe ikakhulu zii-Phe, Leu kunye nee-Val residues kummandla we-transmembrane. Ezi residues zifakwe ii-amino acids ze-hydrophobic kunye nee-pigment ze-αβ-14. Ezinye ii-polar residues nazo zinegalelo ekusebenzisaneni, kubandakanya i-hydrogen bond phakathi kwe-W-Thr68 kunye ne-β-Trp42 kumphezulu we-complex cavity (Umfanekiso 3, F kunye no-G). Kumphezulu we-cytoplasm, i-Gln34 ikufutshane neqela le-keto le-αβ-14 carotenoids. Ukongeza, i-molecule ye-n-dodecyl β-d-maltoside (β-DDM) isonjululwe, kwaye umsila wayo we-hydrophobic wolulelwe kumphezulu phakathi kweproteni-W kunye ne-αβ-14, kwaye umsila we-lipid unokuba semzimbeni. Sikwaqaphele ukuba iindawo zesisombululo se-C-terminal zeprotheyini i-W kunye ne-RCH zisondelelene kakhulu, kodwa azikho ngaphakathi komlinganiselo wokwenza ukusebenzisana okuthile (Umfanekiso 1, A kunye no-E). Nangona kunjalo, kusenokubakho ukusebenzisana kwi-amino acids ze-C-terminal ezingasonjululwanga zezi protheyini zimbini, ezinokubonelela ngendlela yokufumana iprotheyini-W ngexesha lokuhlanganiswa kwe-RC-LH114-W complex.
(A) Iproteni-W, ejongene nojongano ne-LH1αβ14 kwimo yekhathuni, inekhonkco elisecaleni elimile okwentonga (elibomvu), eliboniswe kwinxalenye yomzobo we-electrostatic potential (umphezulu ongwevu ocacileyo onomgangatho we-contour we-0.13). (B) Iproteni-W imelwe ngumphezulu onombala we-hydrophobic. Iindawo ze-Polar kunye ne-charged ziboniswa kwi-cyan, iindawo ze-hydrophobic ziboniswa ngombala omhlophe, kwaye iindawo ze-hydrophobic kakhulu ziboniswa ngombala o-orenji. (C kunye no-D) Iproteni-W imelwe kwikhathuni, ulwalathiso lwayo lufana nolwe-(A) (C), kwaye lujikeleziswa yi-180° (D). Ngokwendawo ekulandelelwano, iintsalela ezahluliweyo zamkela i-rainbow color scheme, apho i-N-terminal iluhlaza okwesibhakabhaka kwaye i-C-terminal ibomvu. (E) Iproteni-W kwimbono efanayo ne-(A), kwaye iintsalela kwi-interface yeproteni-W:LH1 zimelwe ziintonga ezineempawu eziqhotyoshelweyo. (F) Iproteni-W ijikeleziswa ngama-90° xa kuthelekiswa ne-(E) kunye ne-LH1αβ14 kumfanekiso wekhathuni, kwaye xa kuthelekiswa neentsalela ze-interface kumfanekiso webha. Iintsalela ezijingayo ezivela kwi-beta polypeptide ziphawulwe. I-cofactor iboniswa njengebha ehambelana nombala woMfanekiso 1, i-β-DDM ebolileyo iboniswe ngombala ongwevu, kwaye ioksijini iboniswa ngombala obomvu. (G) Umbono kwi-(F) ujikeleziswa ngama-180°, kunye neentsalela ezibalaseleyo ze-alpha polypeptide ephawulwe.
IProtein-W ithatha indawo ye-αβ heterodimer (eye-15 kuMfanekiso 1F), ngaloo ndlela ithintela ukuvalwa kwe-loop kunye nokuthambekisa ii-heterodimer ezintathu zokuqala ze-αβ. Kwabonwa ukuba i-angle ephezulu yokuthambekela kwe-heterodimer yokuqala ye-αβ-1 xa kuthelekiswa nefilimu eqhelekileyo yayiyi-25° ukuya kwi-29° (Umfanekiso 1, A kunye no-E), eyadalwa yi-inclination ye-2° ukuya kwi-8° ye-αβ-1 kwi-RC A sharp contrast-LH116 (Umfanekiso 1, B kunye no-F). Ii-heterodimer zesibini nezesithathu zithambekele kwi-12° ukuya kwi-22° kunye ne-5° ukuya kwi-10°, ngokulandelelana. Ngenxa yesithintelo se-steric se-RC, ukuthambekela kwe-αβ-1 akubandakanyi isibini sesibini se-αβ (esihambelana ne-16th αβ kuMfanekiso 1F), ngaloo ndlela kwakheka umsantsa ocacileyo kwiringi ye-LH1 (Umfanekiso 1, A kunye no-E). Ngenxa yokunqongophala kwee-heterodimer ezimbini ze-αβ, ezihamba nokulahleka kwee-BChl ezine kunye nee-carotenoids ezimbini, akukho nanye yee-carotenoids ebopha kwi-subunit ejijekileyo ye-αβ-1, nto leyo ebangela indandatho ye-LH114-W equlethe ii-carotenoids ezili-13 ze-Vegetarian kunye nee-BChls ezingama-28. Uqikelelo lwesisombululo sendawo lwee-complexes ezimbini kwiindawo ze-αβ1 ukuya kwi-7 luphantsi kunezo ze-loop ye-LH1 eseleyo, enokubonakalisa i-plasticity yendalo ye-subunit ye-LH1 ecaleni kwendawo ye-RC QB (Umfanekiso 4).
Imifanekiso ye-RC-LH114-W (A kunye no-B) kunye ne-RC-LH116 (C kunye no-D) iboniswa ukusuka kumbono ofanayo ophezulu/umbono osecaleni (A kunye no-B) (A kunye no-C) kunye nomphezulu womngxuma woMfanekiso 1. (B kunye no-D). Amaqhosha anemibala aboniswe ngasekunene.
Enye kuphela enye i-core complex ephawulekayo ene-stoichiometric ratio ye-1:14 yi-Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Nangona kunjalo, i-protein W kunye ne-PufX azinazo ii-homology ezicacileyo, kwaye zinempembelelo ebalulekileyo kwizakhiwo zazo ze-LH1. I-PufX yi-TMH enye ene-N-terminal cytoplasmic domain esebenzisana necala le-cytoplasmic le-RC-H subunit (13) kwindawo ehambelana ne-Rps. palustris LH116αβ-16. I-PufX idala itshaneli yokutshintshiselana kwe-quinone/quinolone phakathi kwe-RC-LH1 kunye ne-cytochrome bcl complex kwaye ikhona kuyo yonke i-Rba. sphaeroides core complex (13). Nangona i-interface ye-monomer-monomer ikwi-Rba. I-sphaeroides RC-LH1-PufX dimer ikwindawo yokubopha yeprotheyini W kwi-RC-LH114-W, kwaye isithuba esibangelwa yiPufX kunye neprotheyini-W sikwindawo efanayo (Umfanekiso S7A). Isithuba kwi-RC-LH114-W sikwahambelana netshaneli ye-quinone (8) yePseudomonas rosea LH1, eyenziwe ziipeptides ezingadibaniyo neprotheyini W okanye iPufX (Umfanekiso S7B). Ukongeza, itshaneli ye-quinone kwi-Blc. I-LH1 eluhlaza e-emerald eyenziwe ngokukhupha i-γ subunit enye (7) ikwindawo efanayo (Umfanekiso S7C). Nangona ilawulwa ziiprotheyini ezahlukeneyo, ukubonakala kwezi channels ze-quinone/quinolol kwindawo efanayo kwi-RC-LH1 complex kubonakala ngathi ngumzekelo wokuvela kwezinto ezidibeneyo, okubonisa ukuba isithuba esidalwe yiprotheyini W sinokusebenza njengetshaneli ye-quinone.
Umsantsa kwi-LH114-W loop uvumela ukwakheka kommandla we-membrane oqhubekayo phakathi kwendawo yangaphakathi ye-RC-LH114-W complex kunye ne-bulk membrane (Umfanekiso 1G), endaweni yokudibanisa ii-domains ezimbini nge-protein pore njengakwiiproteni. I-RC-LH116 complex ifana ne-closed Tch. Needle-like complex (22) (Umfanekiso 1H). Ekubeni ukusasazeka kwe-quinone kwi-membrane kukhawuleza kunokusasazwa nge-proteni channel encinci, i-open LH114-W loop inokuvumela ukujika kwe-RC ngokukhawuleza kune-closed LH116 loop, kwaye ukusasazeka kwe-quinone kwi-RC kunokuthintelwa ngakumbi. Ukuze kuvavanywe ukuba iproteni W iyayichaphazela na i-quinones nge-RC, senze uvavanyo lwe-cytochrome oxidation kwi-concentration ethile ye-ubiquinone 2 (UQ2) (i-analogue ye-natural UQ10 enomsila omfutshane we-isoprene) (Umfanekiso 2E). Nangona ubukho be-quinone etyheli buthintela ukuqinisekiswa ngokuchanekileyo kwe-Michaelis constant ebonakalayo (RC-LH114-W kunye ne-RC-LH116 zifanelekile kwi-0.2±0.1μM kunye ne-0.5±0.2μM, ngokulandelelanayo), izinga eliphezulu le-RC-LH114-W (4.6±0.2 e-RC-1 s-1) likhulu ngama-28±5% kune-RC-LH116 (3.6±0.1 e-RC-1 s-1).
Ekuqaleni siqikelele ukuba i-protein-W ikhona malunga ne-10% ye-core complex (16); apha, amazinga okuhlala kweeseli zokukhula ezikhanyayo kancinci, ezikhanyayo eziphakathi, kunye nezikhanyayo eziphezulu yi-15±0.6%, 11±1% kunye ne-0.9±0.5, ngokulandelelana % (Umfanekiso 2F). Uthelekiso lobungakanani lwe-mass spectrometry lubonise ukuba ukongezwa kwe-histidine tag akuzange kunciphise ubuninzi be-protein-W xa kuthelekiswa neentlobo ze-wild-type (P = 0.59), ngoko ke la manqanaba ayisiyonto ye-artifact ye-modified protein-W (Umfanekiso S10). Nangona kunjalo, oku kuhlala okuphantsi kwe-protein-W kwi-RC-LH1 complex kunokuvumela ezinye ii-RC ukuba zijike ngesantya esikhawulezileyo, ngaloo ndlela kuncitshiswe ukutshintshiselana okucothayo kwe-quinone/quinolone kwi-RC-LH116 complex. Siqaphele ukuba izinga lokuhlala elinokukhanya okuphezulu alihambelani nedatha yakutshanje ye-transcriptomics, ebonisa ukuba ukubonakaliswa kwe-pufW gene kuyanda phantsi kokukhanya okunamandla (Umfanekiso S11) (23). Umahluko phakathi kokubhalwa kwe-pufW kunye nokufakwa kwe-protein-W kwi-RC-LH1 complex kuyadida kwaye kunokubonakalisa ukulawulwa okuntsonkothileyo kweproteni.
Kwi-RC-LH114-W, ii-6 cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) kunye nee-29 β-DDM molecules zabelwe kwaye zafakwa imodeli kuzo ii-6 CDLs, 24 POPCs, 2 POPGs kunye ne-12 βDDMs. RC-LH116 (Umfanekiso 5, A kunye no-B). Kwezi zakhiwo zimbini, i-CDL iphantse ibekwe kwicala le-cytoplasmic le-complex, ngelixa i-POPC, i-POPG kunye ne-β-DDM ikakhulu zibekwe kwicala le-luminal. Ii-molecule ezimbini ze-lipid kunye ne-detergent zazahlulwe kummandla we-αβ-1 ukuya kwi-αβ-6 we-RC-LH114-W complex (Umfanekiso 5A), kwaye ezintlanu zazahlulwe kummandla olinganayo we-RC-LH116 (Umfanekiso 5B). Kufunyenwe ii-lipids ezingaphezulu kwelinye icala le-complex, ikakhulu i-CDL, eziqokelelene phakathi kwe-RC kunye ne-αβ-7 ukuya kwi-αβ-13 (Umfanekiso 5, A kunye no-B). Ezinye ii-lipids kunye nee-detergents ezisombululwe ngokwesakhiwo zifumaneka ngaphandle kwendandatho ye-LH1, kwaye ii-acyl chains ezisombululwe kakuhle zanda phakathi kwee-subunits ze-LH1, ezibizwa ngokuba yi-β-DDM kwi-RC-LH114-W, kwaye zichazwa njenge-β-DDM kwi-RC A umxube we-β-DDM kunye ne-POPC-LH116. Iindawo ezifanayo ze-chelating lipids kunye nee-detergents kwisakhiwo sethu zibonisa ukuba ziindawo zokubopha ezifanelekileyo ngokwempilo (Umfanekiso S12A). Iindawo zee-molecules ezifanayo kwi-Tch nazo zinokuhambelana okuhle. Zithambile kwaye ziTrv. I-Strin 970 RC-LH1s (Umfanekiso S12, B ukuya ku-E) (9, 12) kunye neentsalela ze-hydrogen-bonding zeqela le-lipid head zibonise ulondolozo oluhle kakhulu kwi-sequence alignment (Umfanekiso S13), nto leyo ebonisa ukuba i-Conserved CDL ebopha kwi-RC (24), ezi ndawo zinokugcinwa kwi-RC-LH1 complex.
(A kunye no-B) Iipeptide ze-RC-LH114-W (A) kunye ne-RC-LH116 (B) zimelwe ziikhathuni, kwaye imibala imelwe ziintonga, kusetyenziswa isikimu sombala esikuMfanekiso 1. IiLipids ziboniswe ngombala obomvu, kwaye iisepha ziboniswe ngombala ongwevu. I-UQ ebotshelelwe kwiindawo ze-RC QA kunye ne-QB ityheli, ngelixa i-UQ ehlukanisiweyo iluhlaza okwesibhakabhaka. (C kunye no-D) Iimbono ezifanayo nezika-(A) kunye no-(B), kunye neelipids ezishiyiweyo. (E ukuya ku-G) Umbono owandisiweyo we-Q1(E), Q2(F) kunye ne-Q3(G) evela kwi-RC-LH116, kunye neetyathanga ezisecaleni ezichaphazela omnye nomnye. Iibhondi ze-hydrogen ziboniswa njengemigca emnyama eneedayimani.
Kwi-RC-LH116, zombini i-RC QA kunye ne-QB UQ, ezithatha inxaxheba ekudlulisweni kwee-electron kwinkqubo yokwahlulahlula itshaja, ziyabola kwiindawo zazo zokubopha. Nangona kunjalo, kwi-RC-LH114-W, i-QB quinone ayikasonjululwa kwaye kuya kuxoxwa ngayo ngokweenkcukacha ngezantsi. Ukongeza kwi-QA kunye ne-QB quinones, iimolekyuli ezimbini ze-UQ ezi-chelated (eziphakathi kweeringi ze-RC kunye ne-LH1) zabelwe kwisakhiwo se-RC-LH114-W ngokweentloko zazo ezisonjululwe kakuhle (ezikwi-Q1 kunye ne-Q2, ngokulandelelana). Umfanekiso 5C). Iiyunithi ezimbini ze-isoprene zabelwe kwi-Q1, kwaye imephu yoxinano isombulula imisila epheleleyo ye-isoprene eyi-10 ye-Q2. Kwisakhiwo se-RC-LH16, iimolekyuli ezintathu ze-UQ10 ezi-chelated (Q1 ukuya kwi-Q3, Umfanekiso 5D) zasonjululwa, kwaye zonke iimolekyuli zinoxinano olucacileyo kulo lonke umsila (Umfanekiso 5, D ukuya ku-G). Kwizakhiwo ezimbini, iindawo zamaqela eentloko ze-quinone ze-Q1 kunye ne-Q2 zinokuhambelana okuhle kakhulu (Umfanekiso S12F), kwaye zidibana kuphela ne-RC. I-Q1 ikwindawo yokungena kwe-W gap ye-RC-LH114-W (Umfanekiso 1G kunye no-5, C, D kunye no-E), kwaye i-Q2 ikufutshane nendawo yokubopha ye-QB (Umfanekiso 5, C, D) kunye no-F). Iintsalela ze-L-Trp143 kunye ne-L-Trp269 ezigciniweyo zisondele kakhulu kwi-Q1 kunye ne-Q2 kwaye zibonelela ngokusebenzisana okunokwenzeka kwe-π-stacking (Umfanekiso 5, E kunye no-F, kunye nomfanekiso S12). I-L-Gln88, 3.0 Å ukusuka kwi-distal oxygen ye-Q1, inika i-hydrogen bond enamandla (Umfanekiso 5E); le ntsalela igcinwe kuzo zonke ii-RC ngaphandle kobudlelwane obukude kakhulu (Umfanekiso S13). I-L-Ser91 ithathelwe indawo yi-Th ngononophelo kwezinye ii-RC ezininzi (Umfanekiso S13), yi-3.8 Angstroms evela kwi-methyl oxygen ye-Q1, kwaye inokubonelela ngeebhondi ze-hydrogen ezibuthathaka (Umfanekiso 5E). I-Q3 ayibonakali inentsebenziswano ethile, kodwa ikwindawo ephobisayo phakathi kwe-RC-M subunit kunye ne-LH1-α subunit 5 ukuya ku-6 (Umfanekiso 5, D kunye no-G). I-Q1, Q2 kunye ne-Q3 okanye ii-quinones ezikufutshane nazo zisonjululwe kwi-Tch. Gentle, Trv. Strain 970 kunye ne-Blc. Isakhiwo se-iris (9, 10, 12) sikhomba kwindawo yokubopha i-quinone encedisayo egciniweyo kwi-RC-LH1 complex (Umfanekiso S12G). Ii-UQ ezintlanu ezibolileyo kwi-RC-LH116 zivumelana kakuhle ne-5.8±0.7 ye-complex nganye echazwe yi-high performance liquid chromatography (HPLC), ngelixa ii-UQ ezintathu ezibolileyo kwi-RC-LH114-W ziphantsi kunexabiso elilinganisiweyo le-6.2±0.3 (Umzobo S14) libonisa ukuba kukho iimolekyuli ze-UQ ezingasonjululwanga kwisakhiwo.
Iipolypeptides ze-pseudo-symmetric L kunye ne-M nganye iqulethe ii-TMH ezintlanu kwaye zenza i-heterodimer edibanisa i-BChl dimer enye, ii-monomers ezimbini ze-BChl, ii-monomers ezimbini ze-bacteriophage (BPh), kunye ne-non-Heme iron enye kunye ne-molecule enye okanye ezimbini ze-UQ10. Ngenxa yokubakho kweebhondi ze-hydrogen kwiqela le-terminal ketone kunye nokuqokelelana kwayo okwaziwayo kwi-Rps, ii-carotenoids zifakwa kwi-M-subunit, ebizwa ngokuba yi-cis-3,4-dehydroorhodopin. Iintlobo (25). Indawo yangaphandle ye-membrane ye-RC-H inamathele kwi-membrane yi-TMH enye. Ulwakhiwo lwe-RC iyonke lufana ne-RC subunit ezintathu zeentlobo ezinxulumeneyo (ezifana ne-Rba). ii-sphaeroides (PDB ID: 3I4D). Ii-macrocycles ze-BChl kunye ne-BPh, umqolo we-carotenoid kunye ne-non-heme iron ziyadibana ngaphakathi koluhlu lwesisombululo sezi zakhiwo, njengoko kunjalo neqela le-UQ10 head kwindawo ye-QA kunye ne-QB quinone kwi-RC-LH116 (Umfanekiso S15).
Ukufumaneka kwezakhiwo ezimbini ze-RC ezinezinga ezahlukeneyo zokuhlala kwindawo ye-QB kunika ithuba elitsha lokuhlola utshintsho oluhambelanayo oluhamba nokubopha kwe-QB quinone. Kwi-complex ye-RC-LH116, i-QB quinone ikwindawo "ekufutshane" ngokupheleleyo (26), kodwa ukwahlulwa kwe-RC-LH114-W akunayo i-QB quinone. Akukho quinone ye-QB kwi-RC-LH114-W, okumangalisayo kuba i-complex iyasebenza, ngaphezu kwe-RC-LH116 complex ene-QB quinone exazululwe ngokwesakhiwo. Nangona iiringi ezimbini ze-LH1 zijikeleza malunga nee-quinone ezintandathu, ezintlanu zixazululwe ngokwesakhiwo kwiringi ye-RC-LH116 evaliweyo, ngelixa ezintathu kuphela zinqunyelwe ngokwesakhiwo kwiringi ye-RC-LH114-W evulekileyo. Olu phazamiseko lwesakhiwo olukhulayo lunokubonisa ukutshintshwa ngokukhawuleza kweendawo ze-RC-LH114-W QB, i-kinetics ye-quinone ekhawulezayo kwi-complex, kunye namathuba akhulayo okuwela i-LH1 loop. Sicebisa ukuba ukungabikho kwe-UQ kwindawo ye-RC QB ye-RC-LH114-W kusenokuba ngumphumo we-complex enzima ngakumbi nesebenzayo ngakumbi, kwaye indawo ye-QB ye-RC-LH114-W ikhe yamiswa ngoko nangoko kwi-UQ turnover. Inqanaba elithile (ukungena kwindawo ye-QB kuvaliwe) libonisa ukwakheka kwalo msebenzi.
Ngaphandle kwe-QB, ukujikeleza okuhambisanayo kwe-L-Phe217 kwindawo engahambelaniyo nokubopha kwe-UQ10, kuba kuya kubangela ukungqubana kwendawo kunye neyunithi yokuqala ye-isoprene yomsila (Umfanekiso 6A). Ukongeza, utshintsho olucacileyo oluphambili lokwakheka lucacile, ngakumbi i-helix de (i-helix emfutshane kwi-loop phakathi kwe-TMH D kunye ne-E) apho i-L-Phe217 itshintshelwa kwi-pocket yokubopha ye-QB kunye nokujikeleza kwe-L-Tyr223 (Umfanekiso 6A) Ukuphula i-hydrogen bond kunye ne-framework ye-M-Asp45 kunye nokuvala umnyango wendawo yokubopha ye-QB (Umfanekiso 6B). I-Helix de pivots kwisiseko sayo, i-Cα ye-L-Ser209 itshintshelwa yi-0.33Å, ngelixa i-L-Val221Cα itshintshelwa yi-3.52Å. Akukho tshintsho lubonakalayo kwi-TMH D kunye ne-E, olungenakuphikiswa kuzo zombini izakhiwo (Umfanekiso 6A). Ngokwazi kwethu, esi sisakhiwo sokuqala kwi-RC yendalo esivala indawo ye-QB. Uthelekiso nesakhiwo esipheleleyo (esibotshelelwe yi-QB) lubonisa ukuba ngaphambi kokuba i-quinone incitshiswe, kufuneka utshintsho oluthile ukuze ingene kwi-quinone. I-L-Phe217 iyajikeleza ukuze yenze intsebenziswano ye-π-stacking neqela lentloko ye-quinone, kwaye i-helix itshintshela ngaphandle, ivumela i-skeleton ye-L-Gly222 kunye ne-side chain ye-L-Tyr223 ukuba yenze inethiwekhi ye-hydrogen bond enesakhiwo se-hydrogen bond esizinzileyo (Umfanekiso 6, A kunye no-C).
(A) Ikhathuni edibanisayo yehologram (uthotho lwe-L, uthotho lwe-orenji/M, i-magenta) kunye nesakhiwo se-apo (grey), apho iintsalela ezibalulekileyo ziboniswa ngendlela yomfanekiso ofana nentonga. I-UQ10 imelwe yibha etyheli. Umgca onamachaphaza ubonisa iibhondi ze-hydrogen ezenziwe kwisakhiwo sonke. (B kunye no-C) Umzobo womphezulu we-apolipoprotein kunye nesakhiwo sendandatho yonke, ubonisa i-oxygen yekeyini esecaleni ye-L-Phe217 eluhlaza okwesibhakabhaka kunye ne-L-Tyr223 ebomvu, ngokulandelelana. I-subunit ye-L iorenji; iisubunit ze-M kunye ne-H azinambala. (D kunye no-E) I-Apolipoprotein (D) kunye ne-RC QB epheleleyo (E) [umbala ngu-(A) ngokulandelelana] kunye ne-Thermophilus thermophilus PSII (luhlaza, luhlaza okwesibhakabhaka kunye ne-quinone yeplastiki; I-PDB ID: 3WU2) Lungelelanisa (58).
Ngequbuliso, nangona izakhiwo ezininzi ze-RCs ezingenayo i-QB ezingenayo i-LH1 zifumaneka, utshintsho olubonakalayo kolu phononongo aluzange lubikwe ngaphambili. Ezi ziquka isakhiwo sokuncipha kwe-QB esivela kwi-Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) kunye ne-Rba. sphaeroides (PDB ID: 1OGV) (29), zonke eziphantse zifane nesakhiwo sazo se-QB iyonke. Ukuhlolwa ngokusondeleyo kwe-3PRC kutyhile ukuba iimolekyuli zesepha ze-LDAO (Lauryl Dimethyl Amine Oxide) zibopha emnyango wesikhundla se-QB, nto leyo enokuthintela ukuhlelwa kwakhona kwisakhiwo esivaliweyo. Nangona i-LDAO ingaboli kwindawo efanayo kwi-1EYS okanye kwi-1OGV, ezi RC zilungiswa kusetyenziswa isepha efanayo kwaye ke ngoko zinokuvelisa isiphumo esifanayo. Ulwakhiwo lwekristale lwe-Rba. I-Sphaeroides RC edityaniswe ne-cytochrome c2 (PDB ID: 1L9B) nayo ibonakala ngathi inendawo ye-QB evaliweyo. Nangona kunjalo, kule meko, ummandla we-N-terminal we-RC-M polypeptide (osebenzisana nendawo yokubopha ye-QB nge-H bond ye-Tyr residue kwi-Q helix) wamkela ukwakheka okungaqhelekanga, kwaye utshintsho lwe-QB conformational alukhange luhlolwe ngakumbi (30). Okuthuthuzelayo kukuba asikaluboni olu hlobo lokuguqulwa kwe-M polypeptide kwisakhiwo se-RC-LH114-W, esiphantse sifane nommandla we-N-terminal we-RC-LH116 RC. Kufuneka kuqatshelwe ukuba emva kokupheliswa kwe-antenna ye-LH1 esekelwe kwi-detergent, ii-apolipoprotein RCs kwi-PDB zasonjululwa, nto leyo eyasusa ii-pools zangaphakathi ze-quinone kunye ne-lipids kwisithuba esiphakathi kwe-RC kunye nomphezulu wangaphakathi weringi ye-LH1 ejikelezileyo (31, 32). I-RC ihlala isebenza kuba igcina zonke ii-cofactors, ngaphandle kwe-quinone ye-QB ebolayo, engazinzile kangako kwaye idla ngokulahleka ngexesha lenkqubo yokulungiselela (33). Ukongeza, kuyaziwa ukuba ukususwa kwe-LH1 kunye ne-lipids yendalo ye-cyclic kwi-RC kunokuba nefuthe kwimisebenzi, efana nobude bexesha le-P+QB-state ehlukaniswe yitshaja (31, 34, 35). Ke ngoko, sicinga ukuba ubukho bendandatho ye-LH1 yendawo ejikeleze i-RC bunokugcina indawo ye-QB "evaliweyo", ngaloo ndlela kugcinwe imeko-bume yendawo kufutshane ne-QB.
Nangona i-apolipoprotein (engenayo i-QB quinone) kunye nesakhiwo esipheleleyo zimele imifanekiso emibini kuphela yokutshintsha kwendawo ye-QB, endaweni yothotho lweziganeko, kukho imiqondiso yokuba ukubopha kunokuvalelwa ukuze kuthintelwe ukuphinda kudityaniswe yi-hydroquinone Ukuthintela ukuthintela i-substrate. Ukusebenzisana kwe-quinolol kunye ne-quinone kufutshane nendawo ye-QB ye-apolipoprotein kunokwahluka, nto leyo ekhokelela ekwaliweni kwayo yi-RC. Kudala kucetyiswa ukuba utshintsho lwesakhiwo ludlala indima ekubopheni nasekuncitshisweni kwe-quinones. Amandla e-RCs aqandisiweyo okunciphisa ii-quinones emva kokulungiswa okumnyama aphazamisekile (36); i-X-ray crystallography ibonisa ukuba lo monakalo ubangelwa yi-QB quinones ebanjwe kwisakhiwo "esikude" malunga ne-4.5 Å ukusuka kwindawo esebenzayo yeproximal (26), 37). Sicebisa ukuba olu lwakhiwo lwe-distal binding luyifoto yesimo esiphakathi phakathi kwe-apolipoprotein kunye nesakhiwo seringi epheleleyo, elandela ukusebenzisana kokuqala ne-quinone kunye nokuvulwa kwendawo ye-QB.
Uhlobo lwe-RC II olufumaneka kwi-PSII complex yebhaktiriya ethile ye-phototrophic kunye ne-cyanobacteria, i-algae kunye nezityalo lunolondolozo lwesakhiwo nokusebenzayo (38). Ulungelelwaniso lwesakhiwo oluboniswe kuMfanekiso 6 (D kunye no-E) lugxininisa ukufana phakathi kwe-PSII RCs kunye nendawo ye-QB ye-bacterial RC complex. Olu thelekiso belusoloko luyimodeli yokufunda iinkqubo ezinxulumene kakhulu zokubopha kunye nokunciphisa i-quinone. Iimpapasho zangaphambili zicebise ukuba utshintsho lwesakhiwo luhamba nokunciphisa i-quinones ye-PSII (39, 40). Ke ngoko, xa kujongwa ulondolozo lwe-RC oluvela kwindalo, le ndlela yokubopha engakhange ibonwe ngaphambili inokusebenza nakwindawo ye-QB ye-PSII RC kwizityalo ze-phototrophic ezineoksijini.
Ii-Rps ΔpufW (ukususwa kwe-pufW okungabhalwanga) kunye ne-PufW-His (i-C-terminal 10x His-tagged protein-W evezwe kwiintlobo zendalo ze-pufW locus). I-palustris CGA009 ichazwe kumsebenzi wethu wangaphambili (16). Ezi ntlobo kunye nomzali we-isogenic wild-type zifunyenwe kwifriji ngokufaka inani elincinci leeseli kwi-PYE (nganye ilitha ye-5 g -1) (igcinwe kwi-LB kwi--80 °C, equlethe i-50% (w/v) glycerol) protein, i-yeast extract kunye ne-succinate) agar [1.5% (w/v)] plate. Ipleyiti yafakwa ebusuku ebumnyameni kubushushu begumbi phantsi kweemeko ze-anaerobic, yaze yakhanyiswa ngokukhanya okumhlophe (~50 μmolm-2 s-1) okubonelelwe yi-OSRAM 116-W halogen bulbs (RS Components, UK) kangangeentsuku ezi-3 ukuya kwezi-5 de kuvele ikoloni enye. Ikoloni enye yasetyenziswa ukugonya i-10 ml ye-M22+ medium (41) eyongezwe yi-0.1% (w/v) casamino acids (ebizwa ngokuba yi-M22 apha). Le nkcubeko yakhuliswa phantsi kweemeko ze-oxygen ephantsi ebumnyameni kwi-34°C ngokushukuma kwi-180 rpm iiyure ezingama-48, kwaye emva koko i-70 ml yenkcubeko yagonya phantsi kweemeko ezifanayo iiyure ezingama-24. Inkcubeko ye-semi-aerobic enomthamo we-1 ml isetyenziselwa ukugonya i-30 ml ye-M22 medium kwibhotile yeglasi ecacileyo ye-30 ml ye-universal screw-top kwaye itshiswe nge-agitation (~50μmolm-2 s-1) iiyure ezingama-48 ngamandla e-sterile magnetic Stirring rod. Emva koko i-30 ml yenkcubeko yagonya malunga nelitha e-1 yenkcubeko phantsi kweemeko ezifanayo, eyathi emva koko yasetyenziswa ukugonya malunga ne-9 litres yenkcubeko ekhanyisiweyo kwi-~200 μmolm-2 s-1 iiyure ezingama-72. Iiseli zivunwe nge-centrifugation kwi-7132 RCF imizuzu engama-30, zaphinda zaxhonywa kwi-~10 ml ye-20 mM tris-HCl (pH 8.0), zaza zagcinwa kwi--20°C de kube kufuneka.
Emva kokunyibilika, yongeza iikristale ze-deoxyribonuclease I (Merck, UK), i-lysozyme (Merck, UK) kunye neepilisi ezimbini ze-Roche holoenzyme protease inhibitor (Merck, UK) kwiiseli ezivuselelweyo. Kwiseli yoxinzelelo lwesiFrentshi engama-20,000 psi (Aminco, USA), iiseli zaphazamiseka izihlandlo ezisi-8 ukuya kwezili-12. Emva kokususa iiseli ezingaphukanga kunye nenkunkuma enganyibilikiyo ngokuyi-centrifugation kwi-18,500 RCF imizuzu eli-15 kwi-4°C, i-membrane yasuswa kwi-lysate enemibala ngokuyi-centrifugation kwi-113,000 RCF iiyure ezi-2 kwi-43,000°C. Lahla iqhekeza elinyibilikayo kwaye uphinde uyi-suspension i-membrane enemibala kwi-100 ukuya kwi-200 ml ye-20 mM tris-HCl (pH 8.0) kwaye uyi-homogenize de kungabikho zi-aggregates ezibonakalayo. I-membrane exhonyiweyo ifakwe kwi-20 mM tris-HCl (pH 8.0) (Anatrace, USA) equlethe i-2% (w/v) β-DDM kangangeyure e-1 ebumnyameni kwi-4°C ngokuxuba ngobunono. Emva koko i-centrifuge kwi-70°C ukuze inyibilikise i-150,000 RCF kwi-4°C kangangeyure e-1 ukuze kususwe izinto eziseleyo ezinganyibilikiyo.
I-membrane enyibilikisayo evela kwi-ΔpufW strain ifakwe kwikholamu yokutshintshana kwe-ion ye-DEAE ye-50 ml ene-column volumes ezintathu (CV) ze-binding buffer [20 mM tris-HCl (pH 8.0) equlethe i-0.03% (w / v) β-DDM]. Hlamba ikholamu ngee-CV binding buffers ezimbini, uze uhlambe ikholamu ngee-binding buffers ezimbini eziqulethe i-50 mM NaCl. I-RC-LH116 complex yahlulwa nge-linear gradient ye-150 ukuya kwi-300 mM NaCl (kwi-binding buffer) kwi-1.75 CV, kwaye i-binding complex eseleyo yahlulwa nge-binding buffer equlethe i-300 mM NaCl kwi-0.5 CV. Qokelela i-absorption spectrum ephakathi kwe-250 kunye ne-1000 nm, gcina i-fraction enomlinganiselo wokufunxa (A880/A280) engaphezulu kwe-1 kwi-880 ukuya kwi-280 nm, yinyibilikise kabini kwi-binding buffer, kwaye usebenzise inkqubo efanayo kwakhona kwikholamu ye-DEAE On purification. Nyibilikisa ii-fractions ngomlinganiselo we-A880/A280 ophezulu kune-1.7 kunye nomlinganiselo we-A880/A805 ophezulu kune-3.0, yenza umjikelo wesithathu wotshintshiselwano lwe-ion, kwaye ugcine ii-fractions ezinomlinganiselo we-A880/A280 ophezulu kune-2.2 kunye nomlinganiselo we-A880/A805 ophezulu kune-5.0. I-complex ecociweyo kancinci ixutywe ukuya kwi-~2 ml kwisihluzi se-centrifugal se-Amicon 100,000 molecular weight cut-off (MWCO) (Merck, UK), yaza yafakwa kwikholamu yokukhupha ubungakanani be-Superdex 200 16/600 (GE Healthcare, US) equlethe i-200 mM NaCl buffer, yaze yasuswa kwi-buffer efanayo kwi-1.5 CV. Qokelela ii-spectra zokufunxa zeqhezu lokukhupha ubungakanani, kwaye ugxininise ii-spectra zokufunxa nge-A880/A280 ratios ngaphezulu kwe-2.4 kunye ne-A880/A805 ratios ngaphezulu kwe-5.8 ukuya kwi-100 A880, kwaye uyisebenzise ngoko nangoko ukulungiselela okanye ukugcina igridi ye-cryo-TEM Gcina kwi--80°C de kube kufuneka.
I-membrane enyibilikisayo evela kwi-PufW-His strain ifakwe kwikholamu ye-20 ml ye-HisPrep FF Ni-NTA Sepharose (20 mM tris-HCl (pH 8.0) equlethe i-200 mM NaCl kunye ne-0.03% (w/w)) kwi-IMAC buffer (GE Healthcare). v) β-DDM]. Ikholamu yahlanjwa ngee-CV ezintlanu ze-IMAC buffer, yaza emva koko ngee-CV ezintlanu ze-IMAC buffer equlethe i-10 mM histidine. I-core complex yasuswa kwikholamu ngee-buffer ezintlanu ze-IMAC equlethe i-100 mM histidine. Iqhezu eliqulethe i-RC-LH114-W complex ligxininiswe ukuya kwi-~10 ml kwi-tank evuselelweyo exhotyiswe nge-Amicon 100,000 MWCO filter (Merck, UK), yaxutywa amaxesha angama-20 nge-binding buffer, yaza yongezwa kwi-25 ml Kwikholamu ye-DEAE Sepharose, ii-CV ezine ezibotshelelwe kwi-buffer zisetyenziswa kwangaphambili. Hlamba ikholam ngee-CV binding buffers ezine, uze ukhuphe i-complex kwii-CVs ezisibhozo kwi-linear gradient ye-0 ukuya kwi-100 mM NaCl (kwi-binding buffer), kunye nee-CVs ezine eziseleyo ezine-100 mM binding buffer. Ii-residual complexes ezikhutshwe kwi-sodium chloride zidibene ne-A880/A280 ratio ephezulu kune-2.4 kunye ne-A880/A805 ratio ephezulu kune-4.6 fractions zixutywe ukuya kwi-~2 ml kwisihluzi se-centrifugal se-Amicon 100,000 MWCO, kwaye zazaliswa nge-1.5 CV IMAC kwangaphambili. I-Buffer equilibrated Superdex 200 16/600 size exclusion column, kwaye emva koko zikhutshwe kwi-buffer efanayo ngaphezulu kwe-1.5 CV. Qokelela iispectra zokufunxa zeenxalenye ezingabandakanywanga ngobukhulu kwaye ugxininise iispectra zokufunxa ngezilinganiso ze-A880/A280 ngaphezulu kwezilinganiso ze-2.1 kunye ne-A880/A805 ngaphezulu kwe-4.6 ukuya kwi-100 A880, ezisetyenziswa ngoko nangoko ekulungiseleleni igridi ye-TEM eqandisiweyo okanye zigcinwe kwi--80°C de kube kuyimfuneko.
Ifriji yokuntywila yeLeica EM GP yasetyenziselwa ukulungiselela iigridi zeTEM ezinobushushu obuphantsi. Le complex yaxutywa kwi-IMAC buffer ukuya kwi-A880 ye-50, yaza emva koko i-5μl yafakwa kwi-QUANTIFOIL 1.2/1.3 copper-coated mesh entsha ekhutshwe yi-glow-discharged carbon (Agar Scientific, UK). Faka igridi kwi-20°C kunye ne-60% yomswakama ohambelanayo imizuzwana engama-30, uze uyicime imizuzwana emi-3, uze uyicime kwi-ethane engamanzi kwi--176°C.
Idatha ye-RC-LH114-W complex irekhodwe kwi-eBIC (Electronic Bioimaging Center) (British Diamond Light Source) nge-microscope yeTitan Krios, esebenza nge-voltage ekhawulezayo ye-300kV, enomlinganiselo oqhelekileyo we-130,000× kunye namandla e- Khetha isithuba se-20 eV. I-Gatan 968 GIF Quantum ene-K2 peak detector isetyenzisiwe ukurekhoda imifanekiso kwimodi yokubala ukuqokelela idatha. Ubungakanani be-pixel obulinganisiweyo yi-1.048Å, kwaye izinga ledosi yi-3.83 e-Å-2s-1. Iqokelele i-movie kwimizuzwana eli-11 kwaye yayahlulahlula yaba ngamacandelo angama-40. Sebenzisa indawo egqunywe yikhabhoni ukuphinda ugxile kwi-microscope, uze uqokelele ii-movie ezintathu ngomngxuma ngamnye. Zizonke, ii-movie ezingama-3130 ziqokelelwe, kunye namaxabiso e-defocus aphakathi kwe--1 kunye ne--3μm.
Idatha ye-RC-LH116 complex iqokelelwe kusetyenziswa imakroskopu efanayo kwi-Asterbury Biostructure Laboratory (iYunivesithi yaseLeeds, e-UK). Idatha iqokelelwe kwimodi yokubala ngokukhulisa i-130 k, kwaye ubungakanani bepikseli bulinganiswe kwi-1.065 Å ngedosi ye-4.6 e-Å-2s-1. Imuvi irekhodwe kwimizuzwana eli-12 yaza yahlulwahlulwa yangamacandelo angama-48. Zizonke, iifilimu ezingama-3359 ziqokelelwe, kunye namaxabiso e-defocus aphakathi kwe--1 kunye ne--3μm.
Lonke ulungiso lwedatha lwenziwa kwipayipi yeRelion 3.0 (42). Sebenzisa iMotioncorr 2 (43) ukulungisa intshukumo yemitha ngokulinganisa idosi, uze usebenzise iCTTFIND 4.1 (44) ukumisela iparameter yeCTF (umsebenzi wokudlulisa umahluko). Iifotomicrographs eziqhelekileyo emva kwezi zigaba zokuqala zokucubungula ziboniswe kuMfanekiso 2. S16. Itemplate yokukhetha ngokuzenzekelayo iveliswa ngokukhetha ngesandla malunga neepikseli ezingama-250 zee-particles ezili-1000 kwifreyimu ye-pixels ezingama-250 kwaye akukho luhlu lwereferensi olunemilinganiselo emibini (2D), ngaloo ndlela lugatya ezo ntlobo zihlangabezana nongcoliseko lwesampuli okanye ezingenazo iimpawu ezibonakalayo. Emva koko, ukhetho oluzenzekelayo lwenziwa kuzo zonke ii-microphotographs, kwaye i-RC-LH114-W yayingama-particles angama-849,359, kwaye i-RC-LH116 complex yayingama-particles angama-476,547. Zonke iinxalenye ezikhethiweyo ziye zadlula kwimijikelo emibini yokuhlelwa kwe-2D okungengo-reference, kwaye emva kokuqhuba ngakunye, iinxalenye ezidibana nendawo yekhabhoni, ungcoliseko lwesampuli, akukho zimpawu zicacileyo okanye iinxalenye ezigqubanayo kakhulu ziyalahlwa, nto leyo ebangela ukuba iinxalenye ezingama-772,033 (90.9%) kunye nama-359,678 (75.5%) zisetyenziswe kudidi lwe-3D lwe-RC-LH114-W kunye ne-RC-LH116 ngokwahlukeneyo. Imodeli yokuqala yesalathiso se-3D yenziwe kusetyenziswa indlela ye-stochastic gradient desccent. Usebenzisa imodeli yokuqala njengesalathiso, iinxalenye ezikhethiweyo zahlulwe zibe ziindidi ezine kwi-3D. Usebenzisa imodeli kweli candelo njengesalathiso, yenza ukucoca kwe-3D kwiinxalenye ezikudidi olukhulu, emva koko sebenzisa isihluzi sokuqala se-15Å esisezantsi ukugubungela indawo ye-solvent, yongeza ii-pixels ezi-6 zemiphetho ethambileyo, kwaye emva koko ucwangcise ii-pixels ukulungisa umsebenzi wokudlulisa i-Gatan K2 peak Modulation we-top detector. Kwiseti yedatha ye-RC-LH114-W, le modeli yokuqala iguqulwe ngokususa uxinano oluqinileyo kwimiphetho yemaski (iqhawukile kwi-core complex density kwi-UCSF Chimera). Iimodeli eziphumayo (izisombululo ze-RC-LH114-W kunye ne-RC-LH116 ziyi-3.91 kunye ne-4.16 Å, ngokulandelanayo) zisetyenziswa njengesalathiso somjikelo wesibini wohlu lwe-3D. Amasuntswana asetyenzisiweyo ahlanganiswe kwiklasi yokuqala ye-3D kwaye awaqulathi unxibelelwano oluqinileyo nommandla. Apho kudityaniswa okanye kukungabikho kweempawu ezicacileyo zesakhiwo. Emva komjikelo wesibini wohlu lwe-3D, udidi olunesisombululo esiphezulu lukhethiwe [Kwi-RC-LH114-W, udidi olunye lungamasuntswana angama-377,703 (44.5%), kwi-RC-LH116, kukho iindidi ezimbini, zizonke zingamasuntswana angama-260,752 (54.7%) , Apho zifana kuphela xa zilungelelaniswe emva kokujikeleza kokuqala ngomahluko omncinci]. Amasuntswana akhethiweyo aphinda akhutshwe kwibhokisi ye-400-pixel aze acocwe nge-3D refined. I-solvent mask iveliswa kusetyenziswa isihluzo sokuqala se-15Å low-pass, i-3 pixel map expansion kunye ne-3 pixel soft mask. Ukusebenzisa i-per-particle CTF refined, i-per-particle motion correction kunye nomjikelo wesibini we-per-particle CTF refined, i-3D refined, i-solvent masking kunye ne-post-processing zenziwa emva kwenyathelo ngalinye ukuze kuphuculwe ngakumbi ubume obuvelayo. Ukusebenzisa ixabiso elinqunyiweyo le-FSC (Fourier Shell Correlation Coefficient) le-0.143, izisombululo zeemodeli zokugqibela ze-RC-LH114-W kunye ne-RC-LH116 yi-2.65 kunye ne-2.80Å, ngokulandelelana. I-FSC curve yemodeli yokugqibela iboniswe kuMfanekiso 2. S17.
Zonke ii-protein sequences zikhutshelwe kwi-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) yasetyenziswa ukwakha imodeli ye-homology ye-RC, equlethe ii-protein sequences ze-RC-L, RC-M kunye ne-RC-H kwaye isakhiwo sekristale se-Rba. I-sphaeroides RC yasetyenziswa njengetemplate (PDB ID: 5LSE) (46). Sebenzisa isixhobo esithi “fit map” kwi-UCSF Chimera ukuze ulungelelanise imodeli evelisiweyo kwimephu (47), uphucule isakhiwo seprotheyini, kwaye udibanise i-cofactor [4×BChl a (igama le-monomer library residue = BCL), 2×BPh a (BPH), uhlobo olunye okanye ezimbini ze-UQ10 (U10), olunye olungeyo-heme iron (Fe) kunye nolunye oluyi-3,4-dihydrohexacarbonylcholine (QAK)] sebenzisa iCoot (48) ukongeza. Ekubeni i-QAK ingafumaneki kwilayibrari ye-monomer, yahlelwa ngokweeparameter kusetyenziswa isixhobo se-eLBOW kwi-PHENIX (49).
Okulandelayo, kwakhiwa i-subunit ye-LH1. Ekuqaleni, isixhobo sokwakha esizenzekelayo kwi-PHENIX (49) sasetyenziswa ukwakha ngokuzenzekelayo inxalenye yolandelelwano lwe-LH1 kusetyenziswa imephu kunye nolandelelwano lweeproteni ze-LH1-α kunye ne-LH1-β njengengeniso. Khetha i-subunit ye-LH1 epheleleyo, uyikhuphe kwaye uyilayishe kwi-Coot, yongeza ngesandla ulandelelwano olungekhoyo kuyo, kwaye ucocise isakhiwo sonke ngesandla ngaphambi kokongeza ii-BCls ezimbini (BCL) kunye ne-spirilloxanthin (CRT) [ngokwe-Rps efanelekileyo Uxinano lwe-complex ye-LH1 kunye nomxholo we-carotenoid owaziwayo. Iintlobo (17)]. Khuphela i-subunit epheleleyo ye-LH1, kwaye usebenzise i-UCSF Chimera "Docking Map Tool" ukuya kwi-dock kwindawo engeyiyo imodeli ekufutshane yoxinano lwe-LH1, uze uyicocise kwi-Coot; phinda le nkqubo de zonke ii-subunit ze-LH1 zenziwe imodeli. Kwisakhiwo se-RC-LH114-W, ngokukhupha uxinano olungafakwanga kwiCoot, iproteni yahlulwe kwiinxalenye ezingezizo iiproteni eziseleyo kwimephu ye-USCF Chimera kwaye isixhobo se-Autobuild sisetyenziselwa ukuseka imodeli yokuqala, kunye nee-subunits eziseleyo (protein-W) Modeling. Kwi-PHENIX (49). Yongeza naluphi na ulandelelwano olungekhoyo kwimodeli ephumayo kwiCoot (48), uze ucocise ngesandla yonke i-subunit. Uxinano olungafakwanga oluseleyo luhambelana nodibaniso lwee-lipids (ID yelayibrari ye-PDB monomer ye-CDL = CDL, POPC = 6PL kunye ne-POPG = PGT), i-β-DDM detergent (LMT) kunye neemolekyuli ze-UQ10 (U10). Sebenzisa i-PHENIX optimization (49) kunye ne-manual optimization kwiCoot (48) ukuze ugqibelelise imodeli yokuqala epheleleyo de izibalo zemodeli kunye nomgangatho obonakalayo wokulingana zingenakuphuculwa ngakumbi. Okokugqibela, sebenzisa i-LocScale (50) ukuze ulole imephu yendawo, uze wenze eminye imijikelo emininzi yokumodela uxinano olungafakwanga kunye nokulungiswa okuzenzakalelayo kunye nokwesandla.
Iipeptides, ii-cofactors kunye nezinye ii-lipids kunye nee-quinones ezibekwe ngaphakathi kobuninzi bazo ziboniswe kwiMifanekiso 1 kunye no-2. S18 ukuya kwi-S23. Ulwazi lwezibalo zemodeli yokugqibela luboniswe kwiTheyibhile S1.
Ngaphandle kokuba kuchazwe ngenye indlela, ii-UV/Vis/NIR absorption spectra ziqokelelwe kwiCary60 spectrophotometer (e-Agilent, e-USA) kwi-1 nm intervals ukusuka kwi-250 nm ukuya kwi-1000 nm kunye nexesha lokudibanisa le-0.1s.
Nyibilikisa isampuli kwi-quartz cuvette enendlela ye-2 mm ukuya kwi-A880 ye-1, uze uqokelele i-absorption spectrum ephakathi kwe-400 kunye ne-1000 nm. Ii-circular dichroic spectra ziqokelelwe kwi-Jasco 810 spectropolarimeter (eJasco, eJapan) kwi-1 nm intervals phakathi kwe-400 nm kunye ne-950 nm ngesantya se-scan se-20 nm min-1.
I-molar extinction coefficient imiselwa ngokuyixuba i-core complex ibe yi-A880 emalunga nama-50. Nciphisa ivolumu ye-10μl kwi-990μl binding buffer okanye i-methanol, kwaye uqokelele i-absorption spectrum ngoko nangoko ukuze unciphise ukubola kwe-BChl. Umxholo we-BChl wesampuli nganye ye-methanol ubalwe yi-extinction coefficient kwi-771 nm ye-54.8 mM-1 cm-1, kwaye i-extinction coefficient yamiselwa (51). Yahlula i-BChl concentration elinganisiweyo nge-32 (RC-LH114-W) okanye i-36 (RC-LH116) ukuze kuchongwe i-core complex concentration, ethi emva koko isetyenziselwe ukumisela i-absorption spectrum yesampuli efanayo eqokelelwe kwi-buffer Extinction coefficient. parallel. Kuthathwe imilinganiselo emithathu ephindaphindwayo kwisampuli nganye, kwaye i-avareji ye-BChl Qy maximum yasetyenziswa ekubaleni. I-coefficient yokuphela kwe-RC-LH114-W elinganiswe kwi-878 nm yi-3280±140 mM-1 cm-1, ngelixa i-coefficient yokuphela kwe-RC-LH116 elinganiswe kwi-880 nm yi-3800±30 mM-1 cm-1.
I-UQ10 yalinganiswa ngokwendlela eku-(52). Ngamafutshane, i-reverse phase HPLC (RP-HPLC) yenziwe kusetyenziswa inkqubo ye-Agilent 1200 HPLC. Nyibilikisa malunga ne-0.02 nmol ye-RC-LH116 okanye i-RC-LH114-W kwi-50μl ye-50:50 methanol:chloroform equlethe i-0.02% (w/v) ferric chloride, kwaye ufake i-pre-equilibrated Beckman Coulter Ultrasphere ODS 4.6 mm Nyibilikisa kwi-1 ml-1 min-1 kwi-40°C kwi-HPLC solvent (80:20 methanol:2-propanol) kwikholamu ye-×25 cm. Yenza i-isocratic elution kwi-HPLC solvent ukujonga ukufunxwa kwi-275 nm (UQ10), 450 nm (carotenoids) kunye ne-780 nm (BChl) iyure e-1. Incopho kwi-chromatogram engama-275 nm kwimizuzu engama-25.5 yadibaniswa, eyayingenazo ezinye iikhompawundi ezinokubonwa. Indawo edibaniswayo isetyenziselwa ukubala ubungakanani be-molar ye-UQ10 ekhutshiweyo ngokubhekiselele kwi-calibration curve ebalwe kwi-injection yemigangatho ecocekileyo ukusuka kwi-0 ukuya kwi-5.8 nmol (Umfanekiso S14). Isampuli nganye yahlalutywa kwiikopi ezintathu, kwaye impazamo exeliweyo ihambelana ne-SD yomndilili.
Isisombululo esine-RC-LH1 complex enomthamo ophezulu we-Qy wokufunxwa kwe-0.1 salungiswa nge-30 μM ye-cytochrome yentliziyo yehashe encitshisiweyo i-c2 (Merck, UK) kunye ne-0 ukuya kwi-50 μMUQ2 (Merck, UK). Iisampuli ezintathu ze-1-ml zalungiswa kwi-UQ2 concentration nganye zaza zafakwa ebusuku ebumnyameni kwi-4°C ukuqinisekisa ukuziqhelanisa ngokupheleleyo nobumnyama ngaphambi kokulinganisa. Isisombululo safakwa kwi-OLIS RSM1000 modular spectrophotometer exhotyiswe nge-300 nm flame/500 line grating, i-1.24 mm inlet, i-0.12 mm middle kunye ne-0.6 mm outlet slits. I-600 nm long pass filter ibekwe emnyango we-phototube yesampuli kunye ne-reference photomultiplier tube ukuze kuthintelwe ukukhanya okubangela uvuyo. Ukufunxwa kwajongwa kwi-550 nm kunye nexesha lokudibanisa le-0.15 s. Ukukhanya okubangela uvuyo kukhutshwa kwi-880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) ngentambo ye-fiber optic kwi-90% intensity ngokusebenzisa i-DC2200 controller (Thorlabs Ltd., UK) kwaye kukhutshwa kumthombo wokukhanya kwi-engile ye-90° ye. Umqadi wokulinganisa uchasene nesibuko ukuze ubuyisele naluphi na ukukhanya okungazange kufunxwe yisampuli ekuqaleni. Jonga ukufunxwa kwe-10 s ngaphambi kokukhanya kwe-50 s. Emva koko ukufunxwa kwe-10 s kwajongwa ngakumbi imizuzwana engama-60 ebumnyameni ukuze kuvavanywe ubungakanani be-quinolol enciphisa i-cytochrome c23 + ngokuzenzekelayo (jonga uMfanekiso S8 ukuze ufumane idatha eluhlaza).
Idatha icutshungulwe ngokufaka isantya sokuqala esithe ngqo ngaphakathi kwemizuzwana eyi-0.5 ukuya kweyi-10 (kuxhomekeke kuxinzelelo lwe-UQ2) kunye nokulinganisa amazinga azo zonke iisampulu ezintathu kuxinzelelo ngalunye lwe-UQ2. Uxinzelelo lwe-RC-LH1 olubalwe yi-extinction coefficient efanelekileyo lusetyenziselwe ukuguqula isantya sibe yi-catalytic efficiency, edwetshwe kwi-Origin Pro 2019 (OriginLab, e-USA), kwaye lufakwe kwimodeli ye-Michaelis-Menten ukumisela ixabiso le-Km kunye ne-Kcat elibonakalayo.
Kwimilinganiselo yokufunxa okwethutyana, isampuli ye-RC-LH1 yaxutywa ukuya kwi-~2μM kwi-IMAC buffer equlethe i-50 mM sodium ascorbate (Merck, USA) kunye ne-0.4 mM Terbutin (Merck, USA). I-Ascorbic acid isetyenziswa njengomnikeli we-electron ozinikeleyo, kwaye i-tert-butaclofen isetyenziswa njenge-QB inhibitor ukuqinisekisa ukuba umnikeli oyintloko we-RC uhlala enciphile (oko kukuthi, akafakwanga kwi-photooxidized) kuyo yonke inkqubo yokulinganisa. Malunga ne-3 ml yesampuli yongezwa kwiseli ejikelezayo eyenzelwe wena (malunga ne-0.1 m ububanzi, i-350 RPM) enobude bendlela ye-optical ye-2 mm ukuqinisekisa ukuba isampuli kwindlela ye-laser inexesha elaneleyo lokuziqhelanisa nobumnyama phakathi kwee-pulses zokuvuselela. Sebenzisa ii-pulses ze-laser ze-~100-fs ukwandisa inkqubo ye-laser ye-Ti: Sapphire (Spectra Physics, USA) ukuze uvuselele isampuli kwi-880 nm ngesantya sokuphindaphinda se-1 kHz (20 nJ ye-NIR okanye i-100 nJ ye-Vis). Ngaphambi kokuba uqokelele idatha, beka isampuli ekukhanyeni okubangela uvuyo malunga nemizuzu engama-30. Ukuvezwa kuya kubangela ukungasebenzi kwe-QA (mhlawumbi ukunciphisa i-QA kube kanye okanye kabini). Kodwa nceda uqaphele ukuba le nkqubo iyakwazi ukubuyiselwa umva kuba emva kwexesha elide lokuguquguquka kobumnyama, i-RC iya kubuyela kancinci kumsebenzi we-QA. I-Helios spectrometer (Ultrafast Systems, USA) yasetyenziswa ukulinganisa ii-transient spectra ngexesha lokulibaziseka eliyi--10 ukuya kwi-7000 ps​​. Sebenzisa isoftware ye-Surface Xplorer (Ultrafast Systems, USA) ukwahlulahlula iiseti zedatha, uze udibanise kwaye ulungelelanise. Sebenzisa iphakheji yesoftware yeCarpetView (Light Conversion Ltd., Lithuania) ukusebenzisa iseti yedatha edibeneyo ukufumana ii-differential spectra ezinxulumene nokubola, okanye sebenzisa umsebenzi oquka ii-exponents ezininzi kunye nempendulo yesixhobo ukuze ivumelane nokuguquka kwe-single-wavelength spectral kwi-Origin (OriginLab, USA).
Njengoko kukhankanyiwe apha ngasentla (53), kwalungiselelwa ifilimu ye-photosynthetic equlethe i-LH1 complex engenazo zombini i-RC kunye ne-antenna ye-peripheral LH2. I-membrane yaxutywa kwi-20 mM tris (pH 8.0) yaze yafakwa kwi-quartz cuvette enendlela ye-optical ye-2 mm. I-30nJ laser pulse yasetyenziswa ukuvuselela isampuli kwi-540 nm ngexesha lokulibaziseka le--10 ukuya kwi-7000 ps. Cwangcisa iseti yedatha njengoko kuchaziwe kwisampuli ye-Rps.
I-membrane yahluzwa nge-centrifugation kwi-150,000 RCF iiyure ezi-2 kwi-4°C, yaza emva koko ukufunxwa kwayo kwi-880 nm yaphinda yaxhonywa kwi-20 mM tris-HCl (pH 8.0) kunye ne-200 mM NaCl. Nyibilikisa i-membrane ngokuyixuba kancinci kwi-2% (w/v) β-DDM kangangeyure e-1 ebumnyameni kwi-4°C. Isampuli yaxutywa kwi-100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) ukuya kuxinzelelo lweproteni lwe-2.5 mg ml-1 (uhlalutyo lweBio-Rad). Ukucubungula okungakumbi kwenziwe kwindlela epapashwe ngaphambili (54), kwaqala ngokuxutywa kwe-50 μg protein ukuya kwi-50 μl TEAB equlethe i-1% (w/v) sodium laurate (Merck, UK). Emva kokusebenzisa i-sonication imizuzwana engama-60, yancitshiswa nge-5 mM tris(2-carboxyethyl)phosphine (Merck, UK) kwi-37°C imizuzu engama-30. Kwi-S-alkylation, faka isampuli nge-10 mM methyl S-methylthiomethanesulfonate (Merck, UK) kwaye uyifake kwisisombululo se-isopropanol stock se-200 mM imizuzu eli-10 kubushushu begumbi. Ukugaya i-Proteolytic kwenziwe ngokongeza i-2 μg trypsin/endoproteinase Lys-C mix (Promega UK) kwaye yafakwa kwi-37°C iiyure ezi-3. I-laurate surfactant yakhutshwa ngokongeza i-50 μl ethyl acetate kunye ne-10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) kunye ne-vortexing imizuzwana engama-60. Ukwahlulwa kwesigaba kukhuthazwe yi-centrifugation kwi-15,700 RCF imizuzu emi-5. Ngokwemigaqo yomenzi, ikholamu ye-C18 spin (Thermo Fisher Scientific, UK) yasetyenziswa ukufutha ngononophelo nokususa ityuwa kwisigaba esisezantsi esine-peptide. Emva kokomiswa yi-vacuum centrifugation, isampuli yanyibilikiswa kwi-0.5% TFA kunye ne-3% acetonitrile, kwaye i-500 ng yahlalutywa yi-nanoflow RP chromatography edityaniswe ne-mass spectrometry kusetyenziswa iiparameter zenkqubo ezichazwe ngaphambili.
Sebenzisa iMaxQuant v.1.5.3.30 (56) ukuze ufumane iprotheyini kunye nokulinganisa ukuze ukhangele i-Rps. palustris proteome database (www.uniprot.org/proteomes/UP000001426). Idatha ye-mass spectrometry proteomics ifakwe kwi-ProteomeXchange Alliance kusetyenziswa i-PRIDE partner repository (http://proteomecentral.proteomexchange.org) phantsi kwe-dataset identifier PXD020402.
Uhlalutyo lwe-RPLC oludityaniswe ne-electrospray ionization mass spectrometry, i-RC-LH1 complex yalungiswa nge-wild-type Rps. Kusetyenziswa indlela epapashwe ngaphambili (16), uxinaniso lweproteni oluveliswe kwiiseli ze-palustris yayiyi-2 mg ml-1 kwi-20 mM Hepes (pH 7.8), i-100 mM NaCl kunye ne-0.03% (w/v) β- (Bio-Rad analysis) ) DDM. Ngokweprotokholi yomenzi, sebenzisa i-2D purification kit (GE Healthcare, USA) ukuze ukhuphe i-10 μg protein ngendlela yemvula, kwaye unyibilikise i-precipitate kwi-20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile kunye ne-20% (v/v) water. Ii-microliters ezintlanu zahlalutywa yi-RPLC (Dionex RSLC) edityaniswe ne-mass spectrometry (Maxis UHR-TOF, Bruker). Sebenzisa ikholamu yeMabPac 1.2×100 mm (Thermo Fisher Scientific, UK) ukuze wahlukanise kwi-60°C kunye ne-100μlmin -1, ene-gradient ye-85% (v / v) solvent A [0.1% (v / v) FA kunye ne-0.02% (V/v) TFA aqueous solution] ukuya kwi-85%(v/v) solvent B [0.1%(v/v) FA kunye ne-0.02%(v/v) kwi-90%(v/v) acetonitrile TFA] Usebenzisa i-electrospray standard ionization source kunye ne-default parameters ngaphezu kwemizuzu engama-60, i-mass spectrometer ifumana i-100 ukuya kwi-2750 m/z (mass-to-charge ratio). Ngoncedo lwe-ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), dibanisa i-mass spectrum kwii-subunits ze-complex.
Iiseli zikhuliswe iiyure ezingama-72 phantsi kwe-100 ml NF-low (10μMm-2 s-1), ephakathi (30μMm-2 s-1) okanye ephezulu (300μMm-2 s-1). I-M22 medium (i-M22 medium apho i-ammonium sulfate ishiywa khona kwaye i-sodium succinate ithathelwa indawo yi-sodium acetate) kwibhotile ye-screw-top ye-100 ml (23). Kwimijikelo emihlanu yemizuzwana engama-30, ii-0.1 micron glass beads zafakwa ii-beads kwi-volume ratio ye-1:1 ukuze zinyibilikise iiseli kwaye zifakwe efrijini imizuzu emi-5. Izinto ezinganyibilikiyo, ii-cells ezingaphukanga kunye nee-glass beads zasuswa nge-centrifugation kwi-16,000 RCF imizuzu eli-10 kwi-benchtop microcentrifuge. I-membrane yahlulwe kwi-rotor ye-Ti 70.1 ene-100,000 RCF kwi-20 mM tris-HCl (pH 8.0) ene-40/15% (w/w) sucrose gradient kangangeeyure ezili-10.
Njengoko kuchaziwe kumsebenzi wethu wangaphambili, ukufunyanwa kwe-immunodetection ye-His tag kwi-PufW (16). Ngamafutshane, i-purified core complex (11.8 nM) okanye i-membrane equlethe i-RC concentration efanayo (echazwe yi-oxidation esusa i-reduced difference spectrum kwaye ifanise umthwalo kwi-stained gel) kwi-2x SDS loading buffer (Merck, UK) yancitshiswa kabini. Iiproteni zahlulwe kwi-replica 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). I-gel yadaywa nge-Coomassie Brilliant Blue (Bio-Rad, UK) ukuze ilayishe kwaye ibone i-RC-L subunit. Iproteni kwi-gel yesibini yadluliselwa kwi-methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) ukuze i-immunoassay ifumaneke. I-membrane ye-PVDF ivalwe kwi-50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 kunye ne-5% (w / v) ubisi olucoliweyo, yaze yafakwa kwi-anti-His primary antibody (kwi-Dilute the antibody buffer [50 mM tris-HCl (pH 7.6), 150 mM NaCl kunye ne-0.05% (v/v) Tween-20] kwi-1:1000 A190-114A, Bethyl Laboratories, e-USA) kangangeeyure ezi-4. Emva kokuhlamba izihlandlo ezi-3 imizuzu emi-5 kwi-antibody buffer, i-membrane yadityaniswa ne-horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (exutywe ne-1:10,000 kwi-antibody buffer). I-Incubate ukuze ivumele ukubonwa (imizuzu emi-5 emva kokuhlamba kathathu kwi-antibody buffer) kusetyenziswa i-WESTAR ETA C 2.0 chemiluminescence substrate (Cyanagen, Italy) kunye ne-Amersham Imager 600 (GE Healthcare, UK).
Ngokuzoba usasazo lobunzulu bejeli nganye enemibala okanye umzila we-immunoassay, ukudibanisa indawo engaphantsi kwencochoyi kwaye ubale umlinganiselo wobunzulu be-RC-L (ijeli enemibala) kunye neProtein-W (immunoassay), kwi-ImageJ (57) Qhuba umfanekiso. Ezi zilinganiso ziguqulwe zaba yi-molar ratios ngokucinga ukuba umlinganiselo we-RC-L kwiproteni-W kwisampulu ye-RC-LH114-W ecocekileyo yayiyi-1:1 kwaye kulungelelaniswe yonke idatha ngokufanelekileyo.
Ukuze ufumane ezinye izinto ezongezelelweyo kweli nqaku, nceda ujonge http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
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UDavid JK Swainsbury, uPark Qian, uPhilip J. Jackson, uKaitlyn M. Faries, uDariusz M. Niedzwiedzki, uElizabeth C. Martin, uDavid A. Farmer, uLorna A. Malone, uRebecca F. Thompson, uNeil A. Ranson, uDaniel P Canniffe, uMark J. Dickman, uDewey Holten, uChristine Kirmaier, uAndrew Hitchcock, uC. Neil Hunter
Ulwakhiwo olunesisombululo esiphezulu lwe-light trap 1 complex kwiziko lokuphendula lubonelela ngolwazi olutsha malunga ne-quinone dynamics.
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