Nyalo†Likheli lanyalo: OX11 0DE, UK, Sakhiwo Semadayimane, Ipaki Yesayensi Nekusungula YaseHarwell, Dietcote, Oxfordshire, UK, Umtfombo Wekukhanya Kwemadayimane Co.
Sikhungo sekuphendvula sekuvuna kukhanya lokuyinkimbinkimbi 1 (RC-LH1) nguyona ncenye lebalulekile yekukhicita ngekukhanya kwemagciwane labubende ladla kukhanya. Sitfule takhiwo letimbili tesibonakhulu se-cryo-elekthronikhi se-RC-LH1 leyinkimbinkimbi kusuka ku-Rhodopseudomonas palustris. Lesakhiwo sesincumo se-2.65-Å se-RC-LH114-W leyinkimbinkimbi sihlanganisa ema-subunit LH1 langu-14 lazungeze i-RC, lephazanyiswa yi-protein W, kantsi le-complex ngaphandle kwe-protein-W i-RC lephelele letungeletwe yi-RC. Kuvaliwe 16 iyunithi lencane LH1 loop. Kucatsanisa kwaletakhiwo kuniketa lwati ngemandla e-quinone ku-RC-LH1 complex, kufaka ekhatsi tingucuko te-conformational lebetingakacaci ngaphambilini uma kuboshwa i-quinone endzaweni ye-RC QB, kanye nendzawo yetindzawo tekubopha te-quinone letisitako, letisita Kubadlulisela ku-RC. Lesakhiwo lesisezingeni lelisetulu le W protein sivimbela kuvalwa kwe LH1 loop, ngaleyondlela kwenta umzila wekusheshisa kushintja kwe quinone/quinolone.
Emandla laniketwa yi-photosynthesis angagcina cishe konkhe lokuphilako emhlabeni, futsi anemandla lamakhulu e-biotechnology yelilanga. Ngesikhatsi kukhutsatwa iphotosynthesis yemhlabawonkhe, emagciwane labubende ladla kukhanya aphindze akhombise tindlela letehlukene temandla kanye nemandla ekugaya kudla. Tingagwema kukhicita ngekukhanya bese tikhula njengeligciwane lelidla tintfo letihlukahlukene ebumnyameni, tingalungisa initrogen ne carbon dioxide, tikhicite ihydrogen, futsi ticedze emakhompawundi lanephunga (1-3). Kute kuniketwe emandla aletinchubo, kukhanya kumele kuguculwe ngekushesha futsi ngendlela lefanele kube ngemandla emakhemikhali. Lenchubo icala lapho khona le-antenna complex lebamba kukhanya imunya kukhanya bese idlulisela emandla lavalelekile esikhungwini sekuphendvula (RC), ngaleyondlela icala kuhlukaniswa kwekushaja (4 – 7). Iyunithi lesisekelo ye photosynthesis kumagciwane labubende ladla kukhanya akhiwe ngeluhlobo lwe 2 lwe RC, lolutungeletwe yinhlanganisela yekuvuna kukhanya 1 (LH1), leyakha inhlanganisela lesemkhatsini ye RC-LH1. iLH1 yakhiwa ngeluhlu lwema αβ heterodimers lagobile, ngayinye yawo ibopha i-chlorophyll lembili yeligciwane (BChl) i-molecule kanye ne-carotenoid yinye noma letimbili (8-12). I-antenna lelula kakhulu ye-LH1 ine-16 noma 17 αβ heterodimers letungeleta i-RC (9-13) ngendlela levaliwe, kepha kuletinye tindzawo letiyinhloko, ema-peptides lahambako aphazamisa kuchubeka kwe-LH1 letungeletile , Ngaleyo ndlela kukhutsata kusakateka kwe-quinol / quinone kanye ne-cytoch1c1. 13-15). Sitjalo lesibukhwebeletana lesidla kukhanya iRhodopseudomonas (Rps.) silwane lesiyimodeli lesingavisisa emandla kanye nekudluliswa kwema electron lokusekela kukhanya. Isakhiwo sekucala se-crystal se-Rps. Imodeli ye palustris RC-LH1 leyinkimbinkimbi yi RC, itungeletwe ngetikhonkwane letingu 15 te LH1 letingafani, letiphazanyiswa yiphrotheni lengatiwa lebitwa ngekutsi “Iphrotheni W” (14). Iprotheyini-W ngemuva kwaloko yakhonjwa njenge RPA4402, lokuyiprotheyini lengakachazwa le-10.5kDa lene-helices lemitsatfu lebikezelwe ye-transmembrane (TMH) (16). Siphakamisa kutsi siphindze sibite ligama le-rpa4402 lekhoda lifutsi le-rpa402 libe yi-pufW kute lihambisane nendlela yekubita lesetjentiselwa lifutsi lelikhoda i-RC-L, M (pufL, pufM) kanye ne-LH1α, β (pufA, pufB) emayunithi lamancane. Kuyathakazelisa kutsi iprotein-W ikhona kuphela cishe ku 10% we RC-LH1, leveta iRps. palustris ikhicita tintfo letimbili letehlukene te RC-LH1. Lapha, sibika takhiwo te-cryo-EM (cryo-EM) letinesincumo lesiphakeme tetinkimbinkimbi letimbili letiyinhloko, lenye inemaprotheni W kanye ne-14 αβ heterodimers, lenye ngaphandle kwemaprotheni W kanye ne-16 Heterodimer LH1 loop levaliwe. Isakhiwo setfu simelela ushintsho sinyatselo ekucondzeni RC-LH1 inkimbinkimbi Rps. palustris, ngoba sihlatiye linani lelifanako letinhlobo ngamunye futsi sinesisombululo lesenele sekwabela ngalokucacile ipeptide ngayinye kanye nemibala leboshiwe kanye nemafutsa lahlobene kanye nema quinones. Kucatsanisa kwaletakhiwo kukhombisa kutsi lamaprotein lamatsatfu eTMH-W langakatfolakali kunoma nguyiphi lenye indzawo ye RC-LH1 kuze kube ngunyalo akhicita umzila we quinone kwentela kusheshisa kushintja kwe quinone/quinolone. Inani letindzawo tekubopha te lipid kanye ne quinone letigcinwe setikhonjiwe, futsi sivete lushintjo lolusha lwekuhlanganiswa ngemuva kwekuhlanganiswa kwe quinone ne RC, lokungenteka kutsi kulungele i-photosystem II (PSII) RC yetintfo letiphilako letinemoya-mpilo. Lokutfolwe ngitsi kuniketa lwati lolusha ngekuhamba kwekubopha kwe-quinone/quinolone kanye nekushintjana ku-RC-LH1 core complex yeligciwane lelibubende lelidla kukhanya.
Kute kube lula kufundza kabanti ngaletinkimbinkimbi letimbili letitfolakala ku Rps. palustris, sihlukanisa i-RC-LH1 ngayinye ngetindlela tetilwane. Le protein W-leshodako leyinkimbinkimbi (lapha ibitwa ngekutsi yi ΔpufW) yahlanjululwa kusuka kuloluhlobo lolungenayo i-pufW gene (16), futsi yinye kuphela i-RC-LH1 leyinkimbinkimbi lengakhicitwa. Le protein W-lecuketse inkimbinkimbi ikhicitwa luhlobo. Le protein W yaloluhlobo iyaguculwa nge 10x His tag ku C-terminus yayo, kwentela kutsi le protein W-lecuketse complex ingahlanganiswa kahle ne protein W leshoda kakhulu ngekutsi ingahambi insimbi. Lendzawo leyinkimbinkimbi ihlukaniswe ngemphumelelo (16) Kuhlolwa kwe-Affinity (IMAC).
Njengobe kukhonjisiwe kuMfanekiso 1, totimbili letinkimbinkimbi ticuketse emayunithi lamatsatfu lamancane e-RC (RC-L, RC-M kanye ne-RC-H) latungeletwe yi-antenna ye-LH1. Lesakhiwo se-2.80-A salendzawo leyinkimbinkimbi lete emaprotheyini-W sikhombisa 16 αβ heterodimers, leyakha i-LH1 loop levaliwe letungelete ngalokuphelele i-RC, lapha ibitwa ngekutsi yi-RC-LH116 leyinkimbinkimbi. Lesakhiwo se-2.65Å se-protein-W-lecuketse yinkimbinkimbi sine-14-heterodimer LH1 lephazanyiswa yi-protein-W, lapha ibitwa ngekutsi yi-RC-LH114-W.
(A na B) Kumelelwa kwemhlabatsi kwemhlanganiso. (C na D) Ema pigments lahlanganisiwe lavetwa ngetindvuku. (E na F) Letinkimbinkimbi letibonwako kusuka ebusweni be cytoplasmic tinema peptides kanye ne LH1 subunits lemelelwe kumakhathuni, futsi tibhalwe ngekuya kweliwashi kusuka esikhaleni se protein-W [lehambisana nekubhalwa kwe Rba. tintfo letiyinkimbinkimbi (13)]. Ku-LH1-α, umbala we-subunit yemaprotheni umtfubi; ku-LH1-β, umbala we-subunit yemaprotheyini uluhlata sasibhakabhaka; ku protein-W, le protein ibovu; ku RC-H, ngu cyan; ku RC-L, ngu Orange; nge-RC-M, umbala lomnyama. Ema Cofactors amelelwa ngetindvuku, loluhlata lumelele emamolekhyuli eBChl kanye ne BPh a, bukhwebeletana lumelele emamolekhyuli eCarotenoids, bese lolutfubi lumelele emamolekhyuli eUQ10. (G na H) Umbono lokhulisiwe wesikhala seprotheyini-W esigodzini lesilinganako se-RC-LH114-W leyinkimbinkimbi (G) kanye ne-RC-LH116 leyinkimbinkimbi (H). Ema cofactors akhonjiswa ngendlela yekugcwalisa sikhala, i-chelated quinone ikhonjiswa ngeluhlata sasibhakabhaka. Lesikhala se protein-W sigcizelelwa ngulayini loluhlata sasibhakabhaka ku (G), kanye netimbobo letincane lapho khona i-quinone/quinolol isakateka khona kulendandatho ye LH116 igcizelelwa ngulayini lomnyama lonemabala ku (H).
Umfanekiso 1 (A na B) ukhombisa iRC letungeletwe ngetinhlelo letivulekile noma letivaliwe te LH1αβ heterodimers, ngayinye yato ibopha ema BChl lamabili kanye ne carotenoid yinye (Umfanekiso 1, C na D). Tifundvo letindlulileko tikhombise kutsi i-Rps yi-LH1 complex. Endleleni yekukhicita i-spirulina xanthin, letinhlobo ticuketse bantfu labahlangene be-carotenoids (17). Nomakunjalo, iSpiropyrroxanthin nguyona lehamba embili futsi bukhulu bayo buyenetisa. Ngako-ke, sakhetsa kumodela i-spiroxanthin kuto tonkhe tindzawo tekubopha te-LH1. Le alpha kanye ne beta polypeptides ngema TMH langawodvwa lanetindzawo letingaphandle telulwelwesi lolufishane (Umfanekiso 1, A, B, E, na F). Noma nje kucina kwetinsalela letingu 17 ku C-terminus kungabonwa, i-alpha polypeptide yahlukaniswa kusuka ku Met1 kuya ku Ala46 kuto totimbili letinkimbinkimbi. β polypeptide kwancishiswa kusuka ku-Gly4 kuya ku-Tyr52 ku-RC-LH116, futsi kusuka ku-Ser5 kuya ku-Tyr52 ku-RC-LH114-W. Kute kucina kwetinsalela letingu 3 noma letingu 4 te N-terminal noma letingu 13 te C-terminal letabonwa (Umfanekiso S1). Kuhlatiya kwe Mass spectrometry kwe RC-LH1 lehlanganisiwe lelungiselelwe kusuka eluhlobo lwesiganga kwakhombisa kutsi lesigodzi lesilahlekile bekungumphumela wekuhlukana kwe heterologous kwalama peptides (Umfanekiso S1 na S2). I-N-siphelo se-α-Met1 nayo yaphawulwa (f). Lokuhlatiya kwakhombisa kutsi i-α-peptide ifaka tinsalela fMet1 kuya ku-Asp42 / Ala46 / Ala47 / Ala50, bese i-β-peptide ifaka tinsalela Ser2 kuya ku-Ala53, lokuvumelana kahle nemephu yekushisa lokuphansi kwe-EM.
Kuhlanganiswa kwe-α-His29 ne-β-His36 kwenta ema-BChls abukane buso nebuso; i-αβ heterodimer ngayinye ihlangana nabomakhelwane bayo kwenta luphu loluvulekile (RC-LH114-W) noma luphu loluvaliwe (RC-LH116) lolujikeleta i-RC I-exciton lehlanganiswe neluhlu lwemibala (Umfanekiso 1, C na D). Nawucatsanisa ne-877 nm band ye-RC-LH114-W, i-880 nm yekutsatsa kushintja lokubovu kwe-RC-LH116 ngu-3 nm (Umfanekiso 2A). Nomakunjalo, le spectrum ye dichroism leyindingilizi icishe ifane (Umfanekiso 2B), lokukhombisa kutsi noma kunemehluko locacile emkhatsini wema loops lavulekile nalavaliwe, simo sendzawo se BChls siyafana kakhulu. Le redshift yekumunwa ingahle ibe ngumphumela wekuncipha kwekunyakata lokushisako kanye nekusimama lokukhulu ku-loop levaliwe (18, 19), kushintja kwekuhlanganiswa kwe-pigment lokubangelwa yi-loop levaliwe (20, 21), noma inhlanganisela yalemiphumela lemibili (11).
(A) I-Ultraviolet/lebonakalako/lesedvute ne-infrared yekutsatsa, ticoco tayo tibhalwe ngemibala yato lehambisanako futsi tijwayelekile kuya esicongweni se-BPh ku 775 nm. (B) Umbala we-dichroism loyindingilizi lowetayelekile ku-BChl umunya ku-805 nm. (C na D) I-ΔA spectra lekhetsiwe kusuka ku-spectra yekutsatsa sikhatsi-lesixazululwe ye-RC-LH114-W leyinkimbinkimbi (C) kanye ne-RC-LH116 leyinkimbinkimbi (D). Kute kucatsaniswa kancono, onkhe ema-spectra ajwayelekile ku ∆A we −A ku 0.2 ps. (E) Izinga le-cytochrome c2 oxidation ngemuva kwekukhishwa kwemisebe lapho kunetintfo letehlukahlukene te-UQ2 (bona Umfanekiso S8 wemininingwane leluhlata). (F) Emaseli lakhuliswe ngaphansi kwekukhanya lokuphansi, lokusemkhatsini noma lokusetulu (10, 30 noma 300μMm-2 s-1, ngekulandzelana), iphrotheni W kanye ne-RC-L subunits kulendzawo lehlanjululwe kanye nesilinganiso se-membrane lehlukanisiwe. Tincume lizinga lemaprotheyini nge-SDS-polyacrylamide gel electrophoresis kanye ne-immunoassay (bona Umfanekiso S9 ngemininingwane leluhlata). Tincume silinganiso sihlobo RC-LH114-W eyinkimbinkimbi ehlanjululwe. Silinganiso se-RC-L kuya ku-protein-W yalendzawo ngu-1:1.
I-BChls esikhundleni 1 ku-αβ14 loop lekhubatekile ye-RC-LH114-W (Umfanekiso 1, A, C, na-E) isondzelene ne-RC umnikeli loyinhloko (P) ngu-6.8Å kune-BChls lelinganako ku-RC-LH116 (Umfanekiso. 1, B, D, S3); nomakunjalo, i-kinetics yekutsatsa kwesikhashana yaletinkimbinkimbi letimbili ikhombisa kutsi ku-RC-LH114-W na-RC-LH116, sikhatsi sekudluliswa kwemandla ekujabula kusuka ku-LH1 kuya ku-RC ngu-40 ± 4 na-44 ± 3 ps (Umfanekiso 2). , C na D, Umfanekiso S4 kanye neLithebula S2). Kuphindze kubete umehluko lomkhulu ekudluliseleni kwe-elekthronikhi ngaphakatsi kwe-RC (Umfanekiso S5 kanye nembhalo longetiwe lohlobene). Sisola kutsi kuhambisana lokusedvute kwesikhatsi sekudluliswa kwemandla emkhatsini we LH1 ne RC-P kungenca yebanga lelifanako, i-angle kanye nemandla langaba khona e-BChl lenyenti kuma-loop lamabili e-LH1. Kubonakala shengatsi kuhlola iphethini yemandla e-LH1 kuze ufinyelele ebangeni lelincane akusheshi kunekudluliselwa kwemandla ngalokucondzile kusuka etindzaweni letingaphansi kwemandla kuya ku-RC. I-LH1 loop levulekile ku-RC-LH114-W ingaphindze ibe nekunyakata lokungabalulekile kwekushisa ngaphansi kwetimo letiphansi tekushisa tekuhlatiya sakhiwo, futsi kune-αβ14 ring conformation lendze ekushiseni kweligumbi kusuka ebangeni le-pigmentation le-βBChls esikhundleni se-RC 1.
Le RC-LH116 complex icuketse ema-BChls langu-32 kanye nema-carotenoids langu-16, futsi kuhleleka kwayo konkhe kuyafana naloko lokutfolakala ku-Thermochromatium (Tch.) pidpidum [Ibhange Lemininingwane Yemaphrotheni (PDB) ID 5Y5S (12) kanye ne-algae leluhlata (i-PDB ID 6ET5) (10). Ngemuva kwekucondzisa, kuphambuka lokuncane kuphela etikhundleni te-αβ heterodimers kwabonwa, ikakhulukati 1-5, 15, na 16 (Umfanekiso S6). Kuba khona kweprotein-W kunemitselela lebalulekile esakhiweni se-LH1. Ema TMH ayo lamatsatfu ahlanganiswa ngetikhonkwane letimfisha, nge N-terminal eceleni kwe lumen yalenkimbinkimbi kanye ne C-terminal eceleni kwe cytoplasmic (Tibalo 1A na 3, A kuya ku D). Iprotein-W itsandza kakhulu emanti (Umfanekiso 3B), futsi iTMH2 ne TMH3 ihlangana ne LH1αβ-14 kwenta indzawo lehambako (Umfanekiso 3, B na E kuya ku G). Lesikhombisi sakhiwe kakhulu ngetinsalela te Phe, Leu kanye ne Val esifundzeni se transmembrane. Letinsalela tibekwa ngema amino acid latsandza emanti kanye ne αβ-14 yemibala. Letinye tinsalela te polar nato tifaka sandla ekusebentisaneni, kufaka ekhatsi sibopho se hydrogen emkhatsini we W-Thr68 ne β-Trp42 etulu kwemgodzi loyinkimbinkimbi (Umfanekiso 3, F na G). Ebusweni be-cytoplasm, i-Gln34 iseceleni kwelicembu le-keto le-αβ-14. Ngekwengeta, i-n-dodecyl β-d-maltoside (β-DDM) yalungiswa, futsi umsila wayo lotsandza emanti wakhula waya endzaweni lehlanganako emkhatsini wemaprotheyini-W ne-αβ-14, futsi umsila wemafutsa ungahle ube semtimbeni. Siphindze sabona kutsi tifundza tesincumo se-C-terminal se-protein W ne-RCH tisondzelene kakhulu, kepha hhayi ngaphakatsi kwebubanti bekwakha kusebentisana lokutsite (Umfanekiso 1, A na-E). Kepha, kungaba nekusebentisana kuma amino acids e-C-terminal langakacatululwa alamaprotheni lamabili, langaniketa indlela yekufunwa kwemaprotein-W ngesikhatsi sekuhlanganiswa kwe-RC-LH114-W complex.
(A) I-Protein-W, lebukene ne-LH1αβ14 ngendlela yekhathuni, ine-chain leseceleni lemise kwendvuku (lebovu), lekhonjiswe engcenyeni yemdvwebo wemandla e-electrostatic (indzawo lempunga lesobala lenelizinga le-contour le-0.13). (B) I-Protein-W lemelelwa yindzawo lenemibala le-hydrophobic. Tindzawo letise Polar kanye naletishajiwe tikhonjiswa nge cyan, tindzawo letingafuni emanti tikhonjiswa ngembala lomhlophe, bese tindzawo letingafuni emanti kakhulu tikhonjiswa ngembala we orange. (C na D) Iprotein-W lemelelwe kukhathuni, indlela yayo lebonakala ngayo iyafana naleyo lese (A) (C), futsi ijikeletwa nga 180° (D). Ngekuya kwesikhundla ekulandzelaneni, letinsalela letihlukile titsatsa luhlelo lwembala we rainbow, lapho khona iN-terminal iluhlata sasibhakabhaka bese iC-terminal ibovu. (E) I-Protein-W ngendlela lefanako naleyo lekhona ku (A), futsi tinsalela letisesimeni se-protein-W:LH1 timelelwa tindvuku letinemamaki lanamatsiselwe. (F) Iprotein-W ijikeletwa 90 ° sihlobene (E) kanye LH1αβ14 ekumeleleni ikhathuni, kanye sihlobo tinsalela interface ekumeleleni ibha. Letinsalela letilengako letivela kule-beta polypeptide tibhalwe. Le cofactor ikhonjiswa njengebha lehambisana nembala weMfanekiso 1, le β-DDM lebolile ikhonjiswa ngembala lompunga, bese i-oksijini ikhonjiswa ngembala lobovu. (G) Umbono ku (F) ujikeletwa 180°, ngetinsalela letivelele te-alpha polypeptide lebhalwe.
Iprotein-W itsatsa sikhundla se-αβ heterodimer (le-15 kuMfanekiso 1F), ngaleyondlela ivimbela kuvalwa kweluphu kanye nekujika ema-αβ heterodimers lamatsatfu ekucala. Kwabonakala kutsi i-angle lephakeme yekuthambekela kwe-αβ-1 heterodimer yekucala lehambisana nefilimu lejwayelekile bekungu-25° kuya ku-29° (Umfanekiso 1, A na-E), leyakhiwa yi-2° kuya ku-8° yekuthambekela kwe-αβ-1 ku-RC A umehluko lokhaliphile-LH116 andF (Umfanekiso 1, B). Le heterodimers yesibili neyesitsatfu ithambekele ku 12° kuya ku 22° kanye na 5° kuya ku 10°, ngekulandzelana. Ngenca yekuvimbela kwe-steric kwe-RC, kujika kwe-αβ-1 akufaki umbhangqwana wesibili we-αβ (lohambisana ne-16th αβ kuMfanekiso 1F), ngaleyo ndlela kwakha igebe lelicacile eringini ye-LH1 (Umfanekiso 1, A na-E) . Ngenca yekungabikhona kwema heterodimers lamabili eαβ, lahambisana nekulahlekelwa kwema BChl lamane kanye nema carotenoids lamabili, kute ema carotenoids labopha ku αβ-1 subunit lesonteke, lokuholela endandatho ye LH114-W lecuketse ema carotenoids lalishumi nakutsatfu Emifino kanye nema BChls langu 28. Letilinganiso tekusombulula tendzawo taletinkimbinkimbi letimbili letisetigodzini te-αβ1 kuya ku-7 tiphansi kunaleto tayo yonkhe i-LH1 loop, lekungakhombisa bupulasitiki lobukhona be-LH1 subunit leseceleni kwendzawo ye-RC QB (Umfanekiso 4) .
Letitfombe te RC-LH114-W (A na B) kanye ne RC-LH116 (C na D) tikhonjiswa kusuka ekubukeni lokufanako lokusetulu/kubuka eceleni (A na B) (A na C) kanye nendzawo yembobo ye Fig. 1. (B na D). Tikhiye letinemibala tikhonjiswe ngakwesokudla.
Lokunye kuphela lokuyinkimbinkimbi lebalulekile lenesici se-stoichiometric se-1:14 yi-Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Nomakunjalo, iprotein W ne PufX ayinayo i-homology lebonakalako, futsi inemitselela lebalulekile ekwakhiweni kwayo kwe LH1. PufX yi-TMH yinye lenesizinda se-cytoplasmic se-N-terminal lesebentisana neluhlangotsi lwe-cytoplasmic lwe-RC-H subunit (13) esimeni lesihambisana ne-Rps. i-LH116αβ-16. iPufX idala umzila wekushintjana kwe quinone/quinolone emkhatsini we RC-LH1 kanye ne cytochrome bcl leyinkimbinkimbi futsi ikhona kuwo onkhe ema Rba. sphaeroides umgogodla yinkimbinkimbi (13). Nanobe sikhombimsebenti se-monomer-monomer sise-Rba. I-sphaeroides RC-LH1-PufX dimer isendzaweni yekubopha yemaprotheni W ku-RC-LH114-W, futsi igebe lelibangwa yi-PufX kanye nemaprotheni-W lisendzaweni lelinganako (Umfanekiso S7A). Lesikhala ku RC-LH114-W siphindze sihambisane nendlela ye quinone lecatshangelwako (8) ye Pseudomonas rosea LH1, leyakhiwa ngema peptides langahlobene nephrotheni W noma iPufX (Umfanekiso S7B). Ngetulu kwaloko, umzila we-quinone ku-Blc. I-LH1 leluhlata lwe-emerald leyakhiwa ngekungafaki i-γ subunit yinye (7) isesimeni lesifanako (Umfanekiso S7C). Noma nje kulawulwa ngemaprotein lehlukene, kuvela kwaletindlela te quinone/quinolol esimeni lesifanako ku RC-LH1 complex kubonakala ngatsi sibonelo sekutigucukela kwemvelo lokuhlangene, lokukhombisa kutsi igebe lelakhiwe yi protein W lingasebenta njengendlela ye quinone.
Lesikhala ku LH114-W loop sivumela kwakheka kwesifundza se membrane lesichubekako emkhatsini wesikhala sangekhatsi se RC-LH114-W complex kanye ne membrane lenkhulu (Umfanekiso 1G), kunekuhlanganisa letindzawo letimbili ngekusebentisa i-protein pore njengasemaprotein. Le RC-LH116 inkimbinkimbi ifana Tch evaliwe. Inkimbinkimbi lefana nenyaliti (22) (Umfanekiso 1H). Njengoba kusakateka kwe quinone nge membrane kushesha kunekusakateka ngendlela lencane yemaprotein, le LH114-W loop levulekile ingavumela kujika kwe RC ngekushesha kune LH116 loop levaliwe, futsi kusakateka kwe quinone ku RC kungaba nekuvinjelwa kakhulu. Kute sivivinye kutsi ngabe iphrotheni W ithinta kuguculwa kwema-quinones nge-RC, senta luhlolo lwe-cytochrome oxidation ku-ubiquinone 2 (UQ2) letsite (i-analogue ye-UQ10 yemvelo lenemsila lomfishane we-isoprene) (Umfanekiso 2E). Noma nje kuba khona kwe-quinone le-chelated kuvimbela kuncuma lokunembile kwe-Michaelis lebonakalako lehlala njalo (RC-LH114-W ne-RC-LH116 ifanele 0.2 ± 0.1 μM na 0.5 ± 0.2 μM, ngekulandzelana), lizinga leliphakeme le-RC-LH2.4-6-1. ± 5% lenkhulu kune-RC-LH116 (3.6 ± 0.1 e-RC-1 s-1).
Sicale salinganisa kutsi iprotheyini-W ikhona cishe ku-10% we-core complex (16); lapha, emazinga ekuhlala emaseli lakhulako lakhanya kancane, lasemkhatsini, kanye nalakhanya kakhulu ngu 15±0.6%, 11±1% kanye na 0.9±0.5, ngekulandzelana % (Umfanekiso 2F). Kucatsanisa kwesilinganiso se mass spectrometry kwakhombisa kutsi kwengeteka kwe histidine tag akuzange kunciphise buningi be protein-W uma kucatsaniswa netinhlobo tetinhlobo tesiganga (P = 0.59), ngako lamazinga akusiyo intfo leyentiwe ye protein-W leguculwe (Umfanekiso S10). Nomakunjalo, lokuhlala lokuphansi kwe protein-W kulendzawo ye RC-LH1 kungavumela lamanye ema RC kutsi agucuke ngesilinganiso lesisheshako, ngaleyondlela kunciphise kushintjana kancane kwe quinone/quinolone kulendzawo ye RC-LH116. Sibonile kutsi lizinga lekuhlala lelisetulu lekukhanya alihambisani nedatha ye-transcriptomics yakamuva, lekhombisa kutsi kuveta ufuto lwe-pufW kuyakhula ngaphansi kwekukhanya lokucinile (Umfanekiso S11) (23). Umehluko emkhatsini wekubhala kwe pufW kanye nekufakwa kwe protein-W ku RC-LH1 complex kuyadida futsi kungakhombisa kulawulwa lokuyinkimbinkimbi kwe protein.
Ku-RC-LH114-W, 6 cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) kanye nemamolekhyuli langu-29 e-β-DDM ayabelwa futsi akhonjiswa kuyo ema-CDL langu-6, ema-POPC langu-24, ema-POPGDM langu-2 kanye ne-12. RC-LH116 (Umfanekiso 5, A kanye B). Kuletakhiwo letimbili, i-CDL icishe ibe seceleni kwe-cytoplasmic yalenkimbinkimbi, kantsi i-POPC, i-POPG ne-β-DDM ikhona kakhulu eceleni kwe-luminal. Emamolekhyuli lamabili e-lipid kanye nekuhlanta ahlukaniswe esigodzini sase-αβ-1 kuya ku-αβ-6 se-RC-LH114-W complex (Umfanekiso 5A), futsi lasihlanu ahlukaniswe esigodzini lesilinganako se-RC-LH116 (Umfanekiso 5B ). Ema lipids lamanyenti atfolakala ngakulolunye luhlangotsi lwenkimbinkimbi, ikakhulu iCDL, lebutselelwe emkhatsini we RC ne αβ-7 kuya ku αβ-13 (Umfanekiso 5, A na B). Lamanye emafutsa lalungiswe ngesakhiwo kanye netinhlanti tikhona ngaphandle kwendandatho ye-LH1, futsi emaketanga e-acyl lalungiswe kahle akhula emkhatsini wema-subunits e-LH1, lakhetsiwe ngekwesikhashana njenge-β-DDM ku-RC-LH114-W, futsi achazwa njenge-β-DDM ku-RC A ingxube ye-βPOPC-DHDM16. Tikhundla letifanako te-chelating lipids kanye netintfo tekugeza esakhiweni setfu tikhombisa kutsi tindzawo letifanele tekubopha emtimbeni (Umfanekiso S12A). Tikhundla tetinhlayiya letilinganako ku Tch nato tinekuvumelana lokuhle. Mnene futsi Trv. Strain 970 RC-LH1s (Umfanekiso S12, B kuya ku E) (9, 12) kanye netinsalela te-hydrogen-bonding telicembu lenhloko ye-lipid takhombisa kulondvolotwa lokuhle kakhulu ekulandzeleni kwekulandzelana (Umfanekiso S13), lokukhombisa kutsi i-CDL legciniwe lebopha ku-RC (24), letindzawo tingase tibe yi-compCHLx.
(A na B) RC-LH114-W (A) kanye na RC-LH116 (B) ema peptides amelelwa ngemakhathuni, bese ema pigments amelelwa ngetindvuku, kusetjentiswa luhlelo lwemibala ku Figure 1. Emafutsa akhonjiswa ngembala lobovu, bese tintfo tekuhlanta tikhonjiswa ngembala lompunga. I-UQ leboshelwe kumasayithi e-RC QA kanye ne-QB imtfubi, kantsi i-UQ leyehlukile iluhlata sasibhakabhaka. (C na D) Imibono lefanako na (A) na (B), nge lipids leshiywe. (E kuya ku G) Umbono lokhulisiwe we Q1(E), Q2(F) kanye ne Q3(G) kusuka ku RC-LH116, ngemaketanga laseceleni lanemtselela kulomunye nalomunye. Letibopho te-hydrogen tikhonjiswa njengemigca lemnyama lebhalwe ngemabala.
Ku RC-LH116, kokubili iRC QA kanye ne QB UQ, lehlanganyela ekudluliseleni kwe electron enchubeni yekuhlukaniswa kwekushaja, iyabola etindzaweni tabo tekubopha. Kepha, ku RC-LH114-W, iQB quinone ayikacatululwa futsi kutawucocwa ngayo kabanti ngetansi. Ngekwengeta ku QA kanye ne QB quinones, ema molecule lamabili e chelated UQ (lasemkhatsini wemaringi e RC kanye ne LH1) abelwe esakhiweni se RC-LH114-W ngekuya kwemacembu abo enhloko lalungiswe kahle (latfolakala ku Q1 na Q2, ngekulandzelana). sikhala). Umfanekiso 5C). Emayunithi lamabili e-isoprene abelwe ku-Q1, futsi imephu yekucina ixazulula imisila lephelele ye-isoprene lengu-10 ye-Q2. Ekwakhiweni kwe RC-LH116, emamolekhyuli lamatsatfu e-chelated UQ10 (Q1 kuya ku Q3, Umfanekiso 5D) alungiswa, futsi onkhe emamolekhyuli anekucina lokucacile kulo lonkhe umsila (Umfanekiso 5, D kuya ku G). Kuletakhiwo letimbili, tikhundla temacembu enhloko ye quinone ye Q1 na Q2 tinekuvumelana lokuhle kakhulu (Umfanekiso S12F), futsi tisebentisana ne RC kuphela. I-Q1 isendzaweni yekungena kwesikhala se-W se-RC-LH114-W (Umfanekiso 1G na-5, C, D na-E), kantsi i-Q2 isendzaweni lesedvute nendzawo yekubopha ye-QB (Umfanekiso 5, C, D) na-F). Letinsalela te L-Trp143 kanye ne L-Trp269 letigcinwe tisondzelene kakhulu ne Q1 na Q2 futsi tiniketa kusebentisana lokungenteka kwe π-stacking (Umfanekiso 5, E na F, kanye ne Umfanekiso S12). L-Gln88, 3.0 Å kusuka ku-oksijini lekhashane ye-Q1, iniketa sibopho lesicinile se-hydrogen (Umfanekiso 5E); letinsalela tigcinwa kuwo onkhe ema RC ngaphandle kwebudlelwane lobukhashane kakhulu (Umfanekiso S13). L-Ser91 ishintjwa ngendlela yekugcina iThr kulamanye ema RC lamanyenti (Umfanekiso S13), ngu 3.8 Angstroms kusuka ku oksijini ye methyl ye Q1, futsi inganiketa tibopho te hydrogen letibutsakatsaka (Umfanekiso 5E). I-Q3 ayibonakali inekusebentisana lokutsite, kepha isesigodzini lesine-hydrophobic emkhatsini we-RC-M subunit kanye ne-LH1-α subunit 5 kuya ku-6 (Umfanekiso 5, D na G). Q1, Q2 kanye ne Q3 noma ema quinones lasedvute lahlanganisiwe nawo alungisiwe ku Tch. Mnene, Trv. Luhlobo 970 kanye ne Blc. Isakhiwo se-iris (9, 10, 12) sikhomba endzaweni yekubopha ye-quinone lesekelako legcinwe endzaweni ye-RC-LH1 (Umfanekiso S12G). Ema-UQ lasihlanu labolile ku-RC-LH116 avumelana kahle ne-5.8±0.7 ye-UQ ngayinye leyinkimbinkimbi lencunywa yi-chromatography lesetulu lesetulu ye-liquid (HPLC), kantsi lama-UQ lamatsatfu labolile ku-RC-LH114-W aphansi kuneLinani lelilinganisiwe le-6.2±0.3 likhombisa kutsi S4 ikhona. kwakhiwa.
Le polypeptides ye L ne M lefananako ngayinye inema TMH lasihlanu futsi yakha i-heterodimer lehlanganisa i-dimer yinye ye-BChl, ema-monomer lamabili e-BChl, ema-monomer lamabili e-bacteriophage (BPh), kanye nensimbi yinye lengasiyo i-Heme kanye ne-molecule yinye noma letimbili te-UQ10. Ngekubakhona kwetibopho te hydrogen ecenjini le ketone lekugcina kanye nekugcwala kwalo lokwatiwako ku Rps, ema carotenoids afakwa ku M-subunit, lebitwa ngekutsi yi cis-3,4-dehydroorhodopin. Tinhlobo (25). I-domain ye-membrane yangaphandle ye-RC-H iboshelwe kule-membrane yi-TMH yinye. Lesakhiwo se RC sonkhe sifana ne RC lencane lemitsatfu yetinhlobo letihlobene (njenge Rba). ema-ID e-PDB: 3I4D). Ema macrocycles e BChl kanye ne BPh, umgogodla we carotenoid kanye nensimbi lengasiyo i-heme iyahlangana ngaphakatsi kweluhlu lwekusombulula kwaletakhiwo, njengoba kwenta licembu lenhloko ye UQ10 endzaweni ye QA kanye ne QB quinone ku RC-LH116 (Umfanekiso S15).
Kutfolakala kwetakhiwo letimbili te-RC letinemazinga lehlukene ekuhlala endzaweni ye-QB kuniketa litfuba lelisha lekuhlola tingucuko letihambisanako te-conformational letihambisana nekubopha kwe-QB quinone. Kulendzawo leyinkimbinkimbi ye-RC-LH116, i-QB quinone isendzaweni leboshiwe ngalokuphelele “lesedvute” (26), kepha kuhlukaniswa kwe-RC-LH114-W kute i-QB quinone. Kute iQB quinone ku RC-LH114-W, lokumangalisako ngoba lendzawo leyinkimbinkimbi iyasebenta, kakhulu kunendzawo leyinkimbinkimbi ye RC-LH116 lene QB quinone lelungiswe ngesakhiwo. Noma nje lamaringi lamabili e-LH1 a-chelate cishe ema-quinones lasitfupha, lasihlanu alungiswa ngekwesakhiwo kulendandatho levaliwe ye-RC-LH116, kantsi lamatsatfu kuphela lalinganiselwe ngekwesakhiwo kulendandatho ye-RC-LH114-W levulekile . Loku kuphatseka kabi kwesakhiwo lokukhulako kungakhombisa kutsatfwa esikhundleni ngekushesha kwetindzawo te-RC-LH114-W QB, i-quinone kinetics lesheshako kulendzawo leyinkimbinkimbi, kanye nekukhula kwematfuba ekwewela i-LH1 loop. Siphakamisa kutsi kungabi khona kwe-UQ endzaweni ye-RC QB ye-RC-LH114-W kungaba ngumphumela wenkimbinkimbi leyinkimbinkimbi kakhulu futsi lesebenta kakhulu, futsi indzawo ye-QB ye-RC-LH114-W seyivele yafrizwa ekujikeleteni kwe-UQ Sigaba lesitsite (umnyango wendzawo ye-QB sewuvaliwe a le conformation) ref.
Ngaphandle kwe QB, kujikeleta lokuhambisana ne L-Phe217 kuya esimeni lesingahambisani nekubopha kwe UQ10, ngoba kutawubangela kushayisana kwendzawo neyunithi yekucala ye isoprene yemsila (Umfanekiso 6A). Ngetulu kwaloko, tingucuko letibonakalako letiyinhloko te-conformational tiyabonakala, ikakhulukati i-helix de (i-helix lemfisha ku-loop emkhatsini we-TMH D ne-E) lapho i-L-Phe217 ishintjelwa khona ephaketheni lekubopha le-QB kanye nekujikeleta kwe-L-Tyr223 (Umfanekiso 6A ) Kuphula sibopho se-hydrogen ne-QB entrance ye-M- indzawo (Umfanekiso 6B). I-Helix de ijikeleta esigabeni sayo, i-Cα ye-L-Ser209 ishintjwa ngu-0.33Å, kantsi i-L-Val221Cα ishintjwa ngu-3.52Å. Kute tingucuko letibonakalako ku TMH D na E, letibekwa ngetulu kwato totimbili takhiwo (Umfanekiso 6A). Ngekwati kwetfu, lesi sakhiwo sekucala ku RC yemvelo levala indzawo ye QB. Kucatsanisa nesakhiwo lesiphelele (lesiboshiwe se QB) kukhombisa kutsi ngaphambi kwekutsi le quinone incishiswe, lushintjo lwe conformational luyadzingeka kwentela kutsi ingene kule quinone. I-L-Phe217 ijikeleta kwenta kusebentisana kwe-π-kubeka nelicembu lenhloko ye-quinone, bese i-helix iyashintjela ngaphandle, ivumela umgogodla we-L-Gly222 kanye ne-chain leseceleni ye-L-Tyr223 kwenta inethiwekhi yekubopha ye-hydrogen lenesakhiwo sekubopha se-hydrogen lesimile (Umfanekiso 6, A na C).
(A) Ikhathuni lehlanganako ye hologram (L chain, orange/M chain, magenta) kanye ne apo (mphunga) sakhiwo, lapho khona tinsalela letibalulekile tikhonjiswa ngendlela yekumeleleka lokufana nendvuku. UQ10 imelelwa yibha lephuti. Umugca lonemachashazi ukhombisa emabhondi e-hydrogen lakhiwe kuso sonkhe sakhiwo. (B na C) Kumelelwa kwemhlabatsi kwe-apolipoprotein kanye nesakhiwo sonkhe sendandatho, kugcizelela umoya-mpilo we-side chain we-L-Phe217 ngembala loluhlata sasibhakabhaka kanye ne-L-Tyr223 ngembala lobovu, ngekulandzelana. I-L subunit i-wolintshi; ema-subunits e-M na-H akafakwa imibala. (D na E) I-Apolipoprotein (D) kanye netindzawo te-RC QB letiphelele (E) [umbala nga (A) ngekulandzelana] kanye ne-Thermophilus thermophilus PSII (luhlata, loluhlata sasibhakabhaka nge-quinone yepulasitiki; ID ye-PDB: 3WU2) Lungisa (58).
Ngekungalindzeleki, noma takhiwo letinyenti te-QB-deficient RCs ngaphandle kwe-LH1 tiyatfolakala, tingucuko te-conformational letibonwe kulolucwaningo atizange tibikwe ngaphambilini. Loku kufaka ekhatsi sakhiwo sekucitsa i-QB kusuka ku-Blc. viridis (ID ye-PDB: 3PRC) (27), i-Tch. tepidum (ID ye-PDB: 1EYS) (28) kanye ne-Rba. sphaeroides (PDB ID: 1OGV) (29), tonkhe leticishe tifane nesakhiwo sato sonkhe se-QB. Kuhlolwa lokusedvute kwe 3PRC kwaveta kutsi emamolekhyuli e-LDAO (Lauryl Dimethyl Amine Oxide) ekuhlanta abopha emnyango wesikhundla se-QB, lokungavimbela kuhlelwa kabusha kube yindlela levaliwe. Noma iLDAO ingaboli endzaweni lefanako ku 1EYS noma ku 1OGV, lama RC alungiselelwe kusetjentiswa sibulalimagciwane lesifanako ngako-ke angakhicita umphumela lofanako. Isakhiwo se-crystal se-Rba. I-Sphaeroides RC lehlanganiswe ne-cytochrome c2 (PDB ID: 1L9B) nayo ibonakala inendzawo levaliwe ye-QB. Kepha, kulesi simo, sifundza se-N-terminal se-RC-M polypeptide (lesebentisana nendzawo yekubopha ye-QB ngesibopho se-H setinsalela te-Tyr ku-Q helix) yamukela kuhambisana lokungekho emvelweni, futsi kushintja kwekuhambisana kwe-QB akuhlolwa kabanti (30 ). Yini lecinisekisako kutsi asikaboni loluhlobo lwekugucuka kwe-M polypeptide esakhiweni se-RC-LH114-W, lokucishe kufane nesigodzi se-N-terminal se-RC-LH116 RC. Kufanele kuphindze kucashelwe kutsi ngemuva kwekucedza i-antenna ye-LH1 lesekelwe ekugezeni, ema-RCs e-apolipoprotein ku-PDB alungiswa, lokucedze emachibi langekhatsi e-quinone kanye ne-lipids esikhaleni lesisemkhatsini we-RC kanye nendzawo lengekhatsi yendandatho ye-LH1 lezungezile (31, 32). I-RC ihlala isebenta ngoba igcina onkhe ema-cofactors, ngaphandle kwe-QB quinone lebolako, lengasimami kakhulu futsi ivame kulahleka ngesikhatsi sekulungisa (33). Ngetulu kwaloko, kuyatiwa kutsi kususwa kwe-LH1 kanye ne-lipids yemvelo lejikeletako kusuka ku-RC kungaba nemtselela emisebenti, njengekuphila lokufinyetiwe kwe-P + QB-state lehlukaniswe ngekushaja (31, 34, 35). Ngakoke, sicabanga kutsi kubakhona kwendandatho yendzawo ye LH1 letungelete iRC kungagcina indzawo ye QB “levaliwe”, ngaleyondlela kugcine simondzawo sendzawo lesedvute ne QB.
Noma i-apolipoprotein (ngaphandle kwe-QB quinone) kanye nesakhiwo lesiphelele simele titfombe letimbili kuphela tekujika kwendzawo ye-QB, kuneluchungechunge lwetintfo letenteka, kunetinkhomba tekutsi kubopha kungavalwa ngesango kuvikela kubopha kabusha nge-hydroquinone Kuvimbela kuvimbela kwe-substrate. Kusebentisana kwe quinolol ne quinone edvute nendzawo ye QB ye apolipoprotein kungaba kwehlukile, lokuholela ekulahlweni kwayo yi RC. Sekuyisikhathi lesidze kuphakanyiswa kutsi tingucuko te conformational tidlala indzima ekubopheni nasekunciphiseni ema quinones. Ikhono le RCs lefriziwe lekunciphisa ema quinones ngemuva kwekujwayela lokumnyama liyakhubateka (36); I-X-ray crystallography ikhombisa kutsi lomonakalo ubangelwa kutsi ema-quinones e-QB avaleleke ngendlela “lekhashane” lecishe ibe ngu-4.5 Å kusuka endzaweni lesebentako lesedvute (26) , 37). Siphakamisa kutsi le conformation yekubopha lekhashane yi-snapshot yesimo lesisemkhatsini emkhatsini we-apolipoprotein kanye nesakhiwo sendandatho lephelele, lelandzela kusebentisana kwekucala ne-quinone kanye nekuvulwa kwesayithi ye-QB.
Loluhlobo lwe II RC lolutfolakala ku PSII complex yemagciwane latsite ladla kukhanya kanye ne cyanobacteria, i-algae kanye netitjalo kunekugcinwa kwesakhiwo kanye nekusebenta (38). Kuhambisana kwesakhiwo lokukhonjiswe kuMfanekiso 6 (D na E) kugcizelela kufana emkhatsini we-PSII RCs kanye nendzawo ye-QB ye-RC complex yeligciwane. Loku kucatsanisa sekuyisikhathi lesidze kuyimodeli yekufundza tinhlelo letihlobene kakhulu tekubopha kwe quinone kanye nekunciphisa. Tincwadzi letidlule tiphakamise kutsi tingucuko te-conformational tihambisana nekuncishiswa kwe-PSII kwe-quinones (39, 40). Ngakoke, uma ucabanga ngekulondvolotwa kwekutigucukela kwemvelo kwe RC, lendlela yekubopha lebeyingakabonwa ngaphambilini ingaphindze isebente endzaweni ye QB ye PSII RC etitjalweni te phototrophic letinemoya-mpilo.
Rps ΔpufW (kucishwa kwe pufW lokungakabhalwa) kanye ne PufW-Yakhe (C-siphelo 10x Yakhe-lefakwe emaprotheyini-W levetwe kusuka ku-locus yemvelo ye-pufW) tinhlobo. palustris CGA009 kwachazwa emsebentini wetfu wangaphambilini (16). Letinhlobo kanye nemtali weluhlobo lwesiganga lwe-isogenic kwatfolakala efrijini ngekutsi kubhalwe linani lelincane lemaseli ku-PYE (litha ngalinye le-5 g -1) (ligcinwe ku-LB ku--80 °C, lecuketse 50% ( w/v) glycerol) emaprotein, imvubelo lekhishwako kanye ne-succinate (%] agar/1)v. Lelipuleti lafutfunyelwa busuku bonkhe ebumnyameni ngesikhatsi sekushisa ekamelweni ngaphansi kwetimo te anaerobic, bese likhanyiswa ngekukhanya lokumhlophe (~50 μmolm-2 s-1) lokuniketwa ngema OSRAM 116-W halogen bulbs (RS Components, UK) emalanga lamatsatfu kuya kulasihlanu kwaze kwavela ikoloni linye. Likoloni linye lasetjentiswa kugoma 10 ml we M22+ losemkhatsini (41) longetiwe nga 0.1% (w/v) we casamino acids (lapha ibitwa ngekutsi yi M22). Lelisiko lakhuliswa ngaphansi kwetimo letiphansi te oxygen ebumnyameni ku 34°C ngekunyakata nga 180 rpm emahora lamashumi lamane nesiphohlongo, bese 70 ml walelisiko yagonywa ngaphansi kwetimo letifanako emahora lamashumi lamabili nakune. Isiko le-semi-aerobic lelinemthamo we-1 ml lisetjentiselwa kugoma 30 ml we-M22 medium ebhodleleni lengilazi le-30 ml le-screw-top lebonakalako bese liyakhanya ngekunyakata (~50μmolm-2 s-1) emahora langu-48 ngemandla emagnetic langakagcini Stirging rod. Ngemuva kwaloko 30 ml welisiko wagonywa nge 1 litre yelisiko ngaphansi kwetimo letifanako, lekwabese kusetjentiswa kugonywa cishe 9 litres welisiko lelikhanyisiwe ku ~200 μmolm-2 s-1 emahora langu 72. Lamangcamuzane avunwa ngekutsi ahlanganiswe ku 7132 RCF imizuzu lengemashumi lamatsatfu, aphindze ahlanganiswe ku ~10 ml we 20 mM tris-HCl (pH 8.0), bese agcinwa ku -20°C kuze kube ngulapho kudzingeka.
Ngemuva kwekuncibilika, faka lamanye emakristalu e-deoxyribonuclease I (Merck, UK), i-lysozyme (Merck, UK) kanye nemaphilisi lamabili eRoche holoenzyme protease inhibitor (Merck, UK) kumaseli laphindze ahlanganisiwe. Kuseli lecindzetelako yaseFrance lene 20,000 psi (Aminco, eMelika), lamaseli aphazamiseka tikhatsi letingu 8 kuya ku 12. Ngemuva kwekususa emaseli langakaphuki kanye nemfucumfucu lengancibiliki ngekutsi kuhlanganiswe ku 18,500 RCF imizuzu lelishumi nesihlanu ku 4°C, le membrane yacindzetelwa kusuka ku lysate lenembala ngekutsi kuhlanganiswe ku 113,000 RCF emahora lamabili ku 43,000°C. Lahla lencenye lencibilikako bese uphindze ufake le membrane lenembala ku 100 kuya ku 200 ml we 20 mM tris-HCl (pH 8.0) bese uyayifana kuze kubete tintfo letibonakalako. Lelucwencwe lolumisiwe lwafutfunyelwa ku 20 mM tris-HCl (pH 8.0) (Anatrace, eMelika) lecuketse 2% (w/v) β-DDM lihora linye ebumnyameni ku 4°C ngekuvuselela lokumnene. Ngemuva kwaloko centrifuge ku 70°C kwentela kuncibilikisa 150,000 RCF ku 4°C lihora linye kwentela kususa tintfo letingancibiliki letisele.
Lelucwencwe loluncibilikisako loluvela esigabeni se-ΔpufW lwasetjentiswa kukholomu yekushintja kwe-ion ye-DEAE Sepharose lengu-50 ml lenemavolumu lamatsatfu ekholomu (CV) ye-buffer yekubopha [20 mM tris-HCl (pH 8.0) lecuketse 0.03% (w / v) β-DDM]. Geza lekholomu ngetintfo letimbili letibophako te CV, bese ugeza lekholomu ngetintfo letimbili letibophako leticuketse 50 mM NaCl. Le RC-LH116 leyinkimbinkimbi yakhishwa ngendlela lesemgceni ye 150 kuya ku 300 mM NaCl (ku buffer yekubopha) ku 1.75 CV, bese lendzawo lesele yekubopha yakhishwa nge buffer yekubopha lecuketse 300 mM NaCl ku 0.5 CV. Hlanganisa le spectrum yekutsatsa emkhatsini wa 250 na 1000 nm, gcina lencenye lenesilinganiso sekutsatsa (A880/A280) lesikhulu kuna 1 ku 880 kuya ku 280 nm, uyihlanjulule kabili ku buffer yekubopha, bese usebentisa inchubo lefanako futsi ku DEAE column On purification. Hlanganisa letincenye letinetilinganiso te A880/A280 letingetulu kwa 1.7 kanye netilinganiso te A880/A805 letingetulu kwa 3.0, yenta umjikeleto wesitsatfu wekushintjana kwe ion, bese ugcina tincenye letinetilinganiso te A880/A280 letingetulu kwa 2.2 kanye netilinganiso te A880/A805 letingetulu kwa.0. Lendzawo lehlanjululwe kancane yagcizelelwa ku ~2 ml ku-Amicon 100,000 yesisindvo se-molecule (MWCO) lesihlungi se-centrifugal (Merck, UK), futsi ilayishwa ku-Superdex 200 16/600 ikholomu yekungafaki (GE Healthcare, US) lecuketse i-buffer ye-200 m thelume, 1.5 I-CV. Hlanganisa ema spectra ekumuncwa kwencenye yekungafakwa kwebukhulu, bese ugcizelela ema spectra ekumuncwa nge A880/A280 ratios ngetulu kwa 2.4 kanye ne A880/A805 ratios ngetulu kwa 5.8 kuya ku 100 A880, bese ngekushesha uyawasebentisa ekulungiseleleni igridi ye cryo-TEM noma kugcina sidzingo until8°C.
Lelucwencwe loluncibilikisako loluvela ku PufW-Luhlobo lwakhe lwasetjentiswa kukholomu ye HisPrep FF Ni-NTA Sepharose lengu 20 ml (20 mM tris-HCl (pH 8.0) lecuketse 200 mM NaCl kanye na 0.03% (w/w)) ku buffer ye IMAC yemphilo ( GE). v) i-β-DDM]. Lekholomu yagezwa ngema CV lasihlanu e IMAC buffer, bese ngema CV lasihlanu e IMAC buffer lecuketse 10 mM histidine. Le nkimbinkimbi leyinhloko yakhishwa kulekholomu ngetintfo letisihlanu te-IMAC leticuketse i-histidine lengu-100 mM. Lengcenye lecuketse le-RC-LH114-W leyinkimbinkimbi igcila ku ~ 10 ml ethangini lelivutsiwe lelifakwe luhlu lwe-Amicon 100,000 MWCO (Merck, UK), lihlanjululwe tikhatsi letingu-20 nge-buffer yekubopha, bese lingetwa ku-25 ml Ku-DEAE Sephaur ku- kusengaphambili. Geza lekholomu ngetintfo letine letibophako te-CV, bese ukhipha lenkimbinkimbi kuma-CV lasiphohlongo ku-gradient lesemgceni we-0 kuya ku-100 mM NaCl (ku-buffer yekubopha), bese ema-CV lamane lasele lacuketse 100 mM we-buffer yekubopha Letinhlanganisela letisele te-chlobin nge-A8de2. ngetulu kwa 2.4 kanye nesilinganiso se A880/A805 lesingetulu kwa 4.6 tincenye betigcizelelwa ku ~ 2 ml ku Amicon 100,000 MWCO centrifugal filter, futsi tigcwaliswe nge 1.5 CV IMAC kusengaphambili Buffer lelinganiselwe i Superdex 200 ihlanganiswe ngetulu kwe 16/600. 1.5 I-CV. Hlanganisa ema spectra ekutsatsa etincenye tesayizi-lekungafakwa bese ugcizelela ema spectra ekutsatsa nge A880/A280 ratios ngetulu kwa 2.1 kanye ne A880/A805 ratios ngetulu kwa 4.6 kuya ku 100 A880, lesetjentiswa ngekushesha ekulungiseleleni igridi ye TEM lefriziwe noma igcinwe ku -80°C.
I-Leica EM GP freezer yekucwilisa yasetjentiswa kulungiselela emagridi e-TEM lashisako laphansi. Lendzawo lehlanganisiwe yancibilikiswa ku IMAC buffer kuya ku A880 ya 50, kwase kutsi 5μl walayishwa ku QUANTIFOIL 1.2/1.3 lesanda kukhanya-lekhishwa inethi yelitfusi lefakwe ikhabhoni (Agar Scientific, UK). Fukamela le grid ku 20°C kanye na 60% wemswakama lohlobene 30 s, bese uyayibhubhisa yomile 3 s, bese uyayicima ku ethane lengemanti ku -176°C.
Idatha ye-RC-LH114-W leyinkimbinkimbi yabhalwa ku-eBIC (Sikhungo Se-Electronic Bioimaging) (Umtfombo Wekukhanya Kwemadayimane aseBrithani) ngesibonakhulu se-Titan Krios, lesisebenta ngemandla lasheshako langu-300kV. I-Gatan 968 GIF Quantum lene-K2 peak umtfombo yasetjentiswa kubhala titfombe ngendlela yekubala kuhlanganisa idatha. Usayizi wephikseli lolinganisiwe ngu-1.048Å, futsi lizinga lemthamo ngu-3.83 e-Å-2s-1. Wabutsela ndzawonye le-movie ngemizuzwana lengu-11 futsi wayihlukanisa yaba tincenye letingu-40. Sebentisa indzawo lefakwe i-carbon kute uphindze ugcizelele sibonakhulu, bese uhlanganisa emabhayisikobho lamatsatfu ngembobo ngayinye. Sekukonkhe, emabhayisikobho langu-3130 ahlanganiswa, ngemanani e-defocus emkhatsini we--1 na-3μm.
Idatha ye RC-LH116 leyinkimbinkimbi yabutfwa kusetjentiswa sibonakhulu lesifanako eLabhorethri Yesakhiwo Setilwane yase Asterbury (eNyuvesi yase Leeds, eUK). Lemininingwane yabutfwa ngendlela yekubala ngekukhulisa lokungu 130 k, futsi linani le pixel lalinganiswa ku 1.065 Å ngemthamo we 4.6 e-Å-2s-1. Le movie yabhalwa ngemizuzwana lengu-12 futsi yahlukaniswa yaba tincenye letingu-48. Sekukonkhe, emafilimu langu 3359 ahlanganiswa, ngemanani e-defocus emkhatsini we -1 na -3μm.
Konkhe kucubungula idatha kwentiwa emgceni we-Relion 3.0 (42). Sebentisa Motioncorr 2 (43) kulungisa kuhamba kwe-beam ngekulinganisa umthamo, bese usebentisa i-CTFFIND 4.1 (44) kutfola ipharamitha ye-CTF (umsebenti wekudluliswa kwekucatsanisa). Ema-photomicrographs lajwayelekile ngemuva kwaletigaba tekucala tekucubungula akhonjiswe kuMfanekiso 2. S16. Lethempulethi yekukhetsa letentekelako yentiwa ngekukhetsa ngesandla emaphikseli langaba ngu 250 etinhlayiya letingu 1000 kuhlaka lwemaphikseli langu 250 futsi kute kuhlukaniswa lokunetigaba letimbili (2D) lokubhekiswe kukho, ngaleyondlela kulahlwa leto tihlukaniso letihlangabetana nekungcola kwesampula noma letite timphawu letibonakalako. Ngemuva kwaloko, kukhetsa lokutentakalelako kwentiwa kuto tonkhe titfombe letincane, futsi i-RC-LH114-W beyitinhlayiya letingu-849,359, bese i-RC-LH116 leyinkimbinkimbi beyiyinhlayiya lengu-476,547. Tonkhe tinhlayiya letikhetsiwe tibe nemijikeleto lemibili yekuhlukaniswa kwe-2D lokungesiko kwekubhekisa, futsi ngemuva kwekugijima ngamunye, tinhlayiya letihlangabetana nendzawo yekhabhoni, kungcoliswa kwesampula, kute timphawu letibonakalako noma tinhlayiya letihlangene ngamandla tiyalahlwa, lokuholela ku-772,033 (90.9%) kanye nekusetjentiswa kwe-359.578% ye-Parles are359. RC-LH114-W kanye ne-RC-LH116 ngekulandzelana. Imodeli yekucala yekubhekisa ye-3D yakhicitwa kusetjentiswa indlela yekwehla kwe-stochastic gradient. Kusebentisa imodeli yekucala njengesikhombo, tinhlayiya letikhetsiwe tihlukaniswe ngetigaba letine ku-3D. Kusebentisa imodeli kulesigaba njengesikhombo, yenta 3D kucolisa tinhlayiya esigabeni lesikhulu, bese usebentisa ekucaleni 15Å phansi-kudlula luhlu kumbonya indzawo solvent, engeza 6 pixels imiphetho ethambile, bese post-incubo emaphikseli kulungisa Gatan K2 transfer function i phezulu Mo. Ku-RC-LH114-W dataset, le modeli yekucala yashintjwa ngekususa buningi lobucinile emaphetselweni emaski (lobucishiwe kusuka kubuningi lobuyinkimbinkimbi lobuyinhloko ku-UCSF Chimera). Letimodeli letiphumako (tincumo te RC-LH114-W kanye ne RC-LH116 ngu 3.91 na 4.16 Å, ngekulandzelana) tisetjentiswa njengesikhombo semzuliswano wesibili wekuhlukaniswa kwe 3D. Letinhlayiya letisetjentisiwe tihlanganiswa tibe sesigabeni sekucala se 3D futsi aticuketsi kuhambisana lokucinile nendzawo. Kuhlangana noma kungabi khona kwetimphawu tesakhiwo letisobala. Ngemuva kwemjikeleto wesibili wekuhlukaniswa kwe-3D, sigaba lesinesisombululo lesiphakeme kakhulu sakhetfwa [Ku-RC-LH114-W, sigaba sinye ngu-377,703 tinhlayiya (44.5%), ku-RC-LH116, kunetigaba letimbili kuphela, letihlanganisa tinhlayiya letingu-260,752 the W.552 kuphela. ngemuva kwekujikeleta kwekucala ngemehluko lomncane]. Letinhlayiya letikhetsiwe tiphindze tikhishwe ebhokisini le-400-pixel bese tiyalungiswa ngekucwengisiswa kwe-3D. Le mask yekuncibilikisa ikhicitwa kusetjentiswa luhlu lwekucala lwe-15Å lwekudlula lokuphansi, kunwebeka kwemephu yemaphikseli lamatsatfu kanye nemaski latsambile emaphikseli lamatsatfu. Kusebentisa kucwengisiswa kwe CTF ngetinhlayiya ngayinye, kulungiswa kwekunyakata kwetinhlayiya ngayinye kanye nemjikeleto wesibili wekucwengisiswa kwe CTF ngetinhlayiya ngayinye, kucwengisiswa kwe 3D, kufihla kwe solvent kanye nekucubungula ngemuva kwekucubungula kwentiwa ngemuva kwesinyatselo ngasinye kwentela kucolisa kakhulu kucubungula lokuphumelako. Kusebentisa i-FSC (I-Fourier Shell Kuhambisana) linani lekusika le-0.143, tincumo temamodeli ekugcina e-RC-LH114-W kanye ne-RC-LH116 ngu-2.65 na-2.80Å, ngekulandzelana. I-FSC curve yemodeli yekugcina ikhonjisiwe kuMfanekiso 2. S17.
Konkhe kulandzelana kwemaprotheyini kulandzelwa ku-UniProtKB: LH1-β (PufB; ID ye-UniProt: Q6N9L5); LH1-α (i-PufA; i-United ID: Q6N9L4); RC-L (I-ID ye-UniProt: O83005); RC-M (PufM; I-ID ye-UniProt: A0A4Z7); RC-H (i-PuhA; I-ID ye-UniProt: A0A4Z9); Iphrotheni-W (I-PufW; I-ID Ye-UniProt: Q6N1K3). SWISS-MODEL (45) yasetjentiswa kwakha imodeli ye-homology ye-RC, lecuketse kulandzelana kwemaprotheni e-RC-L, RC-M kanye ne-RC-H kanye nesakhiwo se-crystal se-Rba. sphaeroides RC yasetjentiswa njengesifanekiso (PDB ID: 5LSE) (46). Sebentisa lithuluzi “lelibalave lelifanele” ku-UCSF Chimera kute lifane nemodeli leyakhiwe kumephu (47), ncono sakhiwo semaprotheni, kanye ne-cofactor [4 × BChl a (ligama letinsalela temtapolwati we-monomer = BCL), 2 × BPh a (BPH), luhlobo lunye noma letimbili te-UQ10 (U10), lunye lolungekho ku-heFme) . 3,4-dihayidroheksakarbonilkholini (QAK)] sebentisa Coot (48) kwengeta. Njengobe i-QAK ingatfolakali emtapweni we-monomer, yabekwa ngekusebentisa lithuluzi le-eLBOW ku-PHENIX (49).
Ngemuva kwaloko, kwakhiwa i-LH1 subunit. Ekucaleni, lithuluzi lekwakha lelizenzakalelayo ku-PHENIX (49) lasetjentiswa kwakha ngekuzenzakalela incenye yekulandzelana kwe-LH1 kusetjentiswa imephu kanye nekulandzelana kwemaprotheni e-LH1-α kanye ne-LH1-β njengekufaka. Khetsa i-LH1 subunit lephelele kakhulu, uyikhiphe bese uyayilayisha ku Coot, ngesandla wengete kulandzelana lokulahlekile kuyo, bese ngesandla ucwengisise sonkhe sakhiwo ngaphambi kwekutsi wengete ema BCls lamabili a (BCL) kanye ne spirilloxanthin (CRT) [ngekuya kwe Rps lefanele Kucina kwe LH1 complex kanye nekucuketfwe kwe carotenoid leyatiwako. Tinhlobo (17)]. Kopisha i-LH1 subunit lephelele, bese usebentisa i-UCSF Chimera “Lithuluzi Lemephu Yekubeka” kufaka endzaweni lesedvute lengasiyo imodeli yekucina kwe-LH1, bese uyayicwengisisa ku-Coot; phindza lenchubo kuze kube ngulapho onkhe ema-subunits e-LH1 asamodelwe. Kulesakhiwo se RC-LH114-W, ngekukhipha buningi lobungakabelwa ku Coot, le protein ihlukaniswa kusuka etincenyeni letisele letingekho emaprotein kumephu ye USCF Chimera bese lithuluzi le Autobuild lisetjentiswa kusungula imodeli yekucala, kanye nema subunits lasele (le protein-W) Modeling. Ku FENIKSI (49). Ngeta noma ngukuphi kulandzelana lokulahlekile kumfanekiso lophumelelako ku Coot (48), bese ngesandla ucwengise yonkhe i-subunit. Lokusele lokungakabelwanga kuhambisana nekuhlanganiswa kwemafutsa (PDB monomer ID ye CDL = CDL, POPC = 6PL kanye ne POPG = PGT), i-β-DDM yekuhlanta (LMT) kanye ne-UQ10 molecules (U10). Sebentisa PHENIX nekusebenta kahle (49) kanye nekusebenta kahle ngesandla ku Coot (48) kuphelela imodeli yekucala lephelele kuze kube ngulapho tibalo temodeli kanye nelizinga lelibonakalako lekufaneleka lingakwati kwentiwa ncono kakhulu. Ekugcineni, sebentisa iLocScale (50) kulola imephu yendzawo, bese wenta leminye imijikeleto leminyenti yekumodela buningi lobungakabelwa kanye nekusebenta kahle lokuzenzakalelayo nalokwentiwa ngesandla.
Ema peptides lalandzelanako, ema cofactors kanye nalamanye ema lipids kanye nema quinones lafakwe ngaphakatsi kwekucina kwawo akhonjiswe ku Tibalo 1 na 2. S18 kuya ku S23. Lwati lwetibalo lwemodeli yekugcina lukhonjiswe kuThebula S1.
Ngaphandle uma kucaciswe ngalenye indlela, le spectra yekutsatsa i-UV/Vis/NIR yabutfwa ku-Cary60 spectrophotometer (Agilent, eMelika) ngesikhatsi se-1 nm kusuka ku-250 nm kuya ku-1000 nm kanye nesikhatsi sekuhlanganiswa se-0.1s.
Hlanganisa lesampula ku cuvette ye quartz ngendlela lengu 2 mm kuya ku A880 ya 1, bese uhlanganisa le spectrum yekutsatsa emkhatsini wa 400 na 1000 nm. Lemibala leyindingilizi ye dichroic yabutfwa ku Jasco 810 (Jasco, eJapan) ngesikhatsi se 1 nm emkhatsini wa 400 nm na 950 nm ngesilinganiso sekuskena se 20 nm min-1.
Le coefficient yekuphela kwe molar incunywa ngekunciphisa le core complex ibe yi A880 lecishe ibe ngu 50. Hlanganisa umthamo we 10μl ku 990μl we buffer yekubopha noma imethanol, bese uhlanganisa le spectrum yekutsatsa ngekushesha kwentela kunciphisa kubola kwe BChl. Lokucuketfwe kwe BChl kwesampula ngayinye ye methanol kwabalwa ngekutsi kuphelele ku 771 nm ku 54.8 mM-1 cm-1, futsi silinganiso sekuphela sancunywa (51). Hlukanisa lokulinganisiwe kwe BChl lokucuketfwe nga 32 (RC-LH114-W) noma 36 (RC-LH116) kwentela kutfola lokucuketfwe lokuyinkimbinkimbi lokuyinhloko, lokubese kusetjentiswa kutfola umbala wekumuncwa kwesampula lefanako lebutfwe ku buffer Extinction coefficient. linganisele. Kutsatfwa tilinganiso letintsatfu letiphindzaphindziwe kusampula ngayinye, futsi silinganiso sekutsatsa kwe BChl Qy lephakeme sasetjentiswa kubala. Silinganiso sekuphela kwe RC-LH114-W lesilinganiswa ku 878 nm ngu 3280 ± 140 mM-1 cm-1, kantsi silinganiso sekuphela kwe RC-LH116 lesilinganiswa ku 880 nm ngu 3800 ± 30 mM-1 cm-1.
UQ10 kwalinganiswa ngendlela ku (52). Ngamafuphi, i-HPLC yesigaba lesibuyela emuva (RP-HPLC) yentiwa kusetjentiswa luhlelo lwe-Agilent 1200 HPLC. Hlanganisa cishe 0.02 nmol we-RC-LH116 noma i-RC-LH114-W ku-50μl ye-50:50 methanol:chloroform lecuketse 0.02% (w/v) we-ferric chloride, bese ujova i-Beckman Coulter Ultrasphere O 40°C ku HPLC incibilikisi (80:20 imethanol: 2-propanol) kukholomu lengu ×25 cm. Yenta kuhlanjululwa kwe isocratic ku HPLC solvent kwentela kugadza kumuncwa ku 275 nm (UQ10), 450 nm (ema carotenoids) kanye na 780 nm (BChl) lihora linye. Lesicoco ku 275 nm chromatogram ngemizuzu lengu 25.5 sahlanganiswa, lebeyingenaletinye tintfo letibonakalako. Lendzawo lehlanganisiwe isetjentiselwa kubala linani le molar le UQ10 lekhishwe ngekubhekisa ku calibration curve lebalwe kusuka ekujovweni kwemazinga lahlantekile kusuka ku 0 kuya ku 5.8 nmol (Umfanekiso S14). Sampula ngayinye yahlaziywa ngekuphindzaphindza lokutsatfu, futsi liphutsa lelibikiwe lihambisana ne-SD yesilinganiso.
Sisombululo lesicuketse iRC-LH1 leyinkimbinkimbi ngekutsatsa kwe Qy lokukhulu kwa 0.1 kwalungiswa nge 30 μM inhlitiyo yemahhashi lencishisiwe ye cytochrome c2 (Merck, UK) kanye na 0 kuya ku 50 μMUQ2 (Merck, UK). Emasampula lamatsatfu langu 1-ml alungiselelwe ku UQ2 ngayinye futsi afutfunyelwa busuku bonkhe ebumnyameni ku 4°C kwentela kucinisekisa kutsi ayagucuka ngalokuphelele ebumnyameni ngaphambi kwekukala. Lesixazululo salayishwa ku-OLIS RSM1000 spectrophotometer lefakwe 300 nm lilangabi/500 umugca wekufaka, 1.24 mm wekungena, 0.12 mm emkhatsini kanye na 0.6 mm wekuphuma. I-600 nm lendze yekuhlunga ibekwa emnyango we-phototube yesampula kanye ne-photomultiplier tube yekubhekisa kukhipha kukhanya kwekujabula. Lokumunwa kwagadvwa ku 550 nm ngesikhatsi sekuhlanganiswa kwe 0.15 s. Lokukhanya kwekujabula kukhishwa kusuka ku-880 nm M880F2 LED (Diode Lekhipha Kukhanya) (Thorlabs Ltd., UK) ngekhebula le-fibre optic ngemandla langu-90% ngekusebentisa umlawuli we-DC2200 (Thorlabs Ltd., UK) futsi kukhishwa emtfonjeni wekukhanya nge-engeli ye-9. Lomsebenti wekukala uphikisana nesibuko kubuyisela noma ngukuphi kukhanya lebekungakatsatfwa ekucaleni yisampula. Gadza kutsatsa 10 s ngaphambi kwekukhanya kwe 50 s. Ngemuva kwaloko kumuncwa kwaphindze kwagadvwa 60 s ebumnyameni kuhlola kutsi i-quinolol inciphisa kangakanani ngekutivelela i-cytochrome c23 + (bona Umfanekiso S8 wemininingwane leluhlata).
Lemininingwane yacutshungulwa ngekufaka lizinga lekucala lelisemgceni ngaphakatsi kwa 0.5 kuya ku 10 s (kuya ngekuhlanganiswa kwe UQ2) kanye nekulinganisa emazinga awo onkhe emasampula lamatsatfu ku UQ2 ngayinye. Lokucuketfwe kwe RC-LH1 lokubalwe ngumlinganiso wekuphela lokulandzelanako kwasetjentiswa kugucula lelizinga libe ngekusebenta kahle kwe catalytic, lokuhlelwe ku Origin Pro 2019 (OriginLab, eMelika), futsi kwafakwa kumfanekiso we Michaelis-Menten kwentela kutfola emanani labonakalako e Km kanye ne Kcat.
Kute kulinganiswe kutsatsa kwesikhashana, isampula ye-RC-LH1 yancibilikiswa yaba ngu ~ 2μM ku-IMAC buffer lecuketse i-50 mM ye-sodium ascorbate (Merck, eMelika) kanye ne-0.4 mM Terbutin (Merck, eMelika). iAscorbic acid isetjentiswa njengemnikeli we-electron yekunikela, bese i-tert-butaclofen isetjentiswa njengesivimbeli se-QB kwentela kucinisekisa kutsi umnikeli lomkhulu we-RC uhlala ancishisiwe (lokusho kutsi, hhayi photooxidized) kuyoyonkhe inchubo yekukala. Cishe 3 ml wesampula yengetwa ku cell lejikeletako ngekwemkhuba (cishe 0.1 m ngebubanti, 350 RPM) ngebudze bendlela yekubuka lengu 2 mm kucinisekisa kutsi lesampula endleleni ye laser inesikhatsi lesenele sekuvumelana nemnyama emkhatsini wema pulses ekujabula. Sebentisa ~ 100-fs emapulsi e-laser kukhulisa luhlelo lwe-laser lwe-Ti: Sapphire (I-Spectra Physics, eMelika) kuze ujabulise isampula ku-880 nm ngesilinganiso sekuphindzaphindza se-1 kHz (20 nJ ye-NIR noma 100 nJ ye-Vis). Ngaphambi kwekutsi uhlanganise idatha, veza lesampula ekukhanyeni kwekujabula cishe imizuzu lengu 30. Kuchayeka kutawubangela kungasebenti kwe-QA (mhlawumbe kunciphise i-QA kanye noma kabili). Kepha sicela wati kutsi lenchubo iyabuyiselwa emuva ngoba ngemuva kwesikhatsi lesidze sekujwayela lokumnyama, i-RC itawubuyela kancane kancane emsebentini we-QA. I-spectrometer ye-Helios (Tinhlelo Letisheshako, eMelika) yasetjentiswa kukala imibala yesikhashana ngesikhatsi sekubambelela -10 kuya ku-7000 ps . Sebentisa isoftware ye-Surface Xplorer (Tinhlelo Letisheshako, eMelika) kute ukhiphe emasethi emininingwane, bese uyahlanganisa bese uyalinganisa. Sebentisa iphakheji yesoftware ye-CarpetView (Light Conversion Ltd., Lithuania) kusebentisa isethi yedatha lehlanganisiwe kutfola ema-spectra lahlukile lahlobene nekubola, noma usebentise umsebenti lofaka ekhatsi ema-exponents lamanyenti ngekuphendvula kwetintfo tekusebenta kute uhambisane nekugucuka kwe-spectra ye-wavelength yinye ku-Origin, USALa).
Njengobe kushiwo ngenhla (53), ifilimu ye-photosynthesis lecuketse i-LH1 complex leshoda kokubili i-RC kanye ne-antenna ye-LH2 lephelele yalungiswa. Lelucwencwe lwancibilikiswa ku 20 mM tris (pH 8.0) bese lulayishwa ku cuvette ye quartz lenendlela yekubuka lengu 2 mm. I-30nJ laser pulse yasetjentiswa kujabulisa isampula ku 540 nm ngesikhatsi sekubambelela se -10 kuya ku 7000 ps. Cubungula isethi yedatha njengoba kuchaziwe ku Rps. pal isampula.
Lelucwencwe lwahlanganiswa ngekutsi luhlanganiswe ku 150,000 RCF emahora lamabili ku 4°C, bese kutsatfwa kwalo ku 880 nm kwaphindze kwafakwa ku 20 mM tris-HCl (pH 8.0) kanye na 200 mM NaCl. Cibilikisa le membrane ngekutsi uyivuse kancane kancane ku 2% (w/v) β-DDM lihora linye ebumnyameni ku 4°C. Lesampula yahlanjululwa ku 100 mM we triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) kuya ekuhlanganisweni kwemaprotheyini langu 2.5 mg ml-1 (kuhlatiya kwe Bio-Rad). Kucubungula lokunye kwentiwa kusuka endleleni leyashicilelwa ngaphambilini (54), kucala ngekuncibilikisa emaprotheni langu 50 μg abe linani le 50 μl le TEAB lecuketse 1% (w / v) sodium laurate (Merck, UK). Ngemuva kwe-sonication ema-60 s, kwancishiswa nge-5 mM tris (2-carboxyethyl) phosphine (Merck, UK) ku-37 ° C imizuzu lengu-30. Ku S-alkylation, fukamela lesampula nge 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) bese uyayifaka kusuka ku 200 mM isopropanol sisombululo sesitoko imizuzu lelishumi ngekushisa kweligumbi. Kugaya kwemaprotein kwentiwa ngekufaka 2 μg we trypsin/endoproteinase Lys-C lehlanganisiwe (Promega UK) bese kufutfunyelwa ku 37°C emahora lamatsatfu. Le laurate lesetulu yakhishwa ngekutsi kufakwe 50 μl we ethyl acetate kanye na 10 μl 10% (v/v) we LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) kanye nekujikeleta emaminitsi langemashumi lasitfupha. Lokuhlukaniswa kwesigaba kwakhutsatwa ngekutsi kusetjentiswe i-centrifugation ku-15,700 RCF imizuzu lengu-5. Ngekuya kwendlela yemkhiciti, ikholomu yekujika ye C18 (Thermo Fisher Scientific, UK) yasetjentiswa kuphefumula ngekucophelela kanye nekususa luswayi esigabeni lesiphansi lesine peptide. Ngemuva kwekomisa ngekutsi kusetjentiswe umkhatsi, lesampula yancibilikiswa ku 0.5% TFA kanye na 3% acetonitrile, futsi 500 ng yahlaziywa nge nanoflow RP chromatography lehambisana ne spectrometry yebuningi kusetjentiswa emapharamitha eluhlelo lachazwe kabanti ngaphambilini.
Sebentisa iMaxQuant v.1.5.3.30 (56) yekukhonjwa kwemaprotheyini kanye nekulinganisa kusesha iRps. imininingwane yemaproteome e-palustris (www. Idatha ye-proteomics ye-mass spectrometry ifakwe ku-ProteomeXchange Alliance ngekusebentisa indzawo yekugcina umlingani we-PRIDE ngaphasi kwesikhombi se-dataset PXD020402.
Kuhlatiya yi-RPLC lehambisana ne-electrospray ye-ionization ye-electrospray, le-RC-LH1 leyinkimbinkimbi yalungiswa kusuka ku-Rps yeluhlobo lwesiganga. Kusebentisa indlela leyashicilelwa ngaphambilini (16), emaprotheni lakhicitwa kumaseli e-palustris bekungu-2 mg ml-1 ku-20 mM Hepes (pH 7.8), 100 mM NaCl kanye na-0.03% (w/v) β- (kuhlatiya kwe-Bio-Rad) ) DDM. Ngekuya kwendlela yemkhiciti, sebentisa ikhithi yekuhlanta ye 2D (i GE Healthcare, eMelika) kukhipha 10 μg wemaprotein ngendlela yekukhipha imvula, bese uncibilikisa lendzawo lecijile ku 20 μl 60% (v / v) we formic acid (FA), 20% (v / v) Acetonitrile/ 20% vter. Ema-microlitre lasihlanu ahlaziywa yi-RPLC (i-Dionex RSLC) lehambisana ne-mass spectrometry (i-Maxis UHR-TOF, i-Bruker). Sebentisa iMabPac 1.2 × 100 mm ikholomu (Thermo Fisher Scientific, UK) yekuhlukanisa ku 60 ° C kanye na 100 μlmin -1, nge gradient ye 85% (v / v) sincibilikisi A [0.1% (v / v) FA kanye na 0.02% (V / v) sisombululo se TFA / v8 a [0.1% (v / v) FA kanye 0.02% (v / v) ku 90% (v / v) i-acetonitrile TFA] Kusebentisa umtfombo we-ionization we-electrospray lojwayelekile kanye nemikhawulo lejwayelekile imizuzu lengetulu kwalengu-60, i-spectrometer yebukhulu itfola 100 kuya ku-2750 m / z (silinganiso sebukhulu-kuya-kushaja). Ngekusitwa yi-exPASy bioinformatics portal ithuluzi le-FindPept (https://web.expasy.org/findpept/), imephu ye-spectrum yebuningi kuya kumayunithi lamancane alendzawo leyinkimbinkimbi.
Lamangcamuzane akhuliswa emahora langemashumi lasikhombisa nakubili ngaphansi kwe 100 ml ye NF-lephansi (10μMm-2 s-1), lesemkhatsini (30μMm-2 s-1) noma lesetulu (300μMm-2 s-1) yekukhanya. M22 emkhatsini (M22 emkhatsini lapho khona i-ammonium sulfate ishiywa khona bese i-sodium succinate itsatfwa sikhundla yi-sodium acetate) ebhodleleni le-100 ml lelisetulu ngesikulufu (23). Emijikelezweni lesihlanu yema 30-s, buhlalu bengilazi lobungu 0.1 micron bebuhlalu ngesilinganiso sevolumu lengu 1:1 kwentela ku lyse emaseli bese bubandza eqhweni imizuzu lesihlanu. Lentfo lengancibiliki, emaseli langakaphuki kanye nebuhlalu bengilazi kwasuswa ngekutsi kuhlanganiswe ku 16,000 RCF imizuzu lelishumi ku microcentrifuge lesebhentjini. Lelucwencwe lwahlukaniswa ku Ti 70.1 rotor nge RCF lengu 100,000 ku 20 mM tris-HCl (pH 8.0) nge 40/15% (w/w) ye sucrose lehambako emahora lalishumi.
Njengobe kuchaziwe emsebentini wetfu wangaphambilini, immunodetection yethegi Yakhe ku-PufW (16). Ngamafuphi, le core complex lehlanjululwe (11.8 nM) noma ulwelwesi lolucuketse lokufanako kwe RC (lokuncunywa yi oxidation kususa spectrum umehluko loncishisiwe kanye nekuhambisana nemtfwalo ku gel lefakwe emabala) ku 2x SDS loading buffer (Merck, UK) lehlanjululwe kabili. Emaprotheyini ahlukaniswe ngendlela lephindzaphindvwako lengu 12% ye bis-tris NuPage gel (i-Thermo Fisher yesayensi, e-UK). Ijeli yagcotjwa nge Coomassie Luhlata Sasibhakabhaka Lokhanyako (Bio-Rad, UK) kulayisha kanye nekubona ngemehlo engcondvo i-RC-L lencane. Leprotheyini lekule jeli yesibili yadluliselwa elucwencweni lwe-polyvinylidene fluoride (i-PVDF) lesebenta nge-methanol (i-Thermo Fisher Scientific, e-UK) kwentela kuhlolwa kwemtimba. Lelucwencwe lwe-PVDF lwavinjelwa ku-50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Emkhatsini-20 kanye ne-5% (w / v) imphuphu yelubisi lolukhishiwe, bese iyafutfunyelwa nge-anti-His antibody leyinhloko (ku-buffer i-mH0p 7.6), 150 mM NaCl kanye na 0.05% (v / v) Emkhatsini-20] ku 1: 1000 A190-114A, Emalabhorethri aseBethyl, eMelika) emahora langu-4. Ngemuva kwekugeza tikhatsi letingu-3 imizuzu lesihlanu ku-buffer yekulwa netifo, lolulwelwesi lwahlanganiswa ne-horseradish peroxidase (Sigma-Aldrich, UK) kulwa ne-horseradish peroxidase (Sigma-Aldrich, UK) kulwa neligundvwane kwesibili (lokuhlanjululwe 1:10,000 ku-buffer yekulwa netifo) Wfukamela kute kuvunyelwe kutfolakala (imizuzu lesihlanu ngemuva kwekugeza lokutsatfu ku-buffer yekulwa ne-antibody EESTAR0). (I-Cyanagen, e-Italy) kanye ne-Amersham Imager 600 (I-GE Yekunakekelwa kweMphilo, e-UK).
Ngekudvweba kusabalalisa kwemandla kwe-gel ngayinye lenebala noma umzila we-immunoassay, kuhlanganisa indzawo lengaphansi kwesicoco bese kubala silinganiso semandla se-RC-L (i-gel lenebala) kanye ne-Protein-W (immunoassay), ku-ImageJ (57) Cubungula sitfombe. Letilinganiso taguculwa taba tilinganiso te molar ngekutsatsa kutsi silinganiso se RC-L kuya ku protein-W kusampula lehlantekile ye RC-LH114-W bekungu 1:1 kanye nekujwayelisa yonkhe idatha isethi ngendlela lefanele.
Kute utfole tintfo letengetiwe talesihloko, sicela ubuke http://advances.sciencemag.org/cgi.
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