Present†Adrɛs naw: OX11 0DE, UK, Daymɔnd Bildin, Harwell Sayns ɛn Inovashɔn Pak, Dietcote, Oxfordshire, UK, Daymɔnd Layt Sɔs Kɔmni, Ltd., Ilɛktronik Bayolojikal Imej Sɛnta.
di riakshכn sεntr layt-havεst kכmpleks 1 (RC-LH1) na di kכr fכtosεntetik kכmכpכnt fכ pכpul fכtotrofik baktri dεm. Wi introdכks tu krayo-εlektrכn maykroskכpi strכkchכ dεm fכ di RC-LH1 kכmpleks frכm Rhodopseudomonas palustris. di 2.65-Å rεsכlushכn strכkchכ fכ di RC-LH114-W kכmpleks kכnsis fכ 14 sכbyunit LH1 lכp dεm we de rawnd RC, we protin W de intarap, we di kכmpleks we nכ gεt protin-W na komplit RC kכmכshכn we RC de rawnd. Klos 16 sabyunit LH1 lɔp. di kכmpεreshכn fכ dεn strכkchכ dεm ya de gi insayt fכ di dinamiks fכ kwinכn na di RC-LH1 kכmpleks, inklud di kכnfכmeshכnal chenj dεm we dεn nכ bin dεtεmin bifo we dεn de biεn kwinכn na di RC QB sayt, εn bak di lכkεshכn fכ כksijεn kvinכn binding sayt dεm, we de εp fכ Pas dεm to RC. di yכŋ strכkchכ fכ di W protin de mek di LH1 lכp nכ kכloz, we de mek wan chanεl fכ aksεlεrayt di kwinכn/kwinכlכn εkshεnj.
Di ɛnaji we fotosintesis de gi kin mek ɔlmost ɔl di layf dɛn we de na di wɔl kɔntinyu fɔ de, ɛn i gɛt bɔku pawa fɔ yuz di solar bayɔteknɔlɔji. pan tap we dεn de protεkt gכl fכtosεntesis, pכpul fכtotrofik baktri dεm de sho difrεn εnεji mכd dεm εn mεtabolik kεpabiliti dεm bak. dεn kin avכyd fכtosεntesis εn gro lεk hεterotrofik baktri dεm na dak, dεn kin fiks naytrojen εn kabכn dayכksayd, prodyuz haydrojεn, εn dεgrad aromatכk kכmpawnd dεm (1-3). Fɔ mek dɛn ebul fɔ gi ɛnaji fɔ dɛn tin ya, layt fɔ chenj kwik ɛn fayn to kemikal ɛnaji. dis prכsεs de stat we di layt-trap εntena kכmpleks de abzכp layt εn tכn di εnεji we dεn trכp to di riakshכn sεntr (RC), we de stat chaj separeshכn (4 – 7). di besik yunit fכ fכtosεntesis insay pכpul fכtotrofik baktri dεm kכmכp fכ tayp 2 RC, we layt-havεst kכmpleks 1 (LH1) de rawnd am, we de fכm di RC-LH1 kכr kכmpleks. LH1 de fכm bay wan εryu fכ kכv αβ hεterodaymεr dεm, εvri wan pan dεm de biεn tu baktriyal klorofil (BChl) a mכlikul dεm εn wan כ tu karotenoyd dεm (8-12). di simpul LH1 antena de kכnsis fכ 16 כ 17 αβ hεterodaymεr dεm we de rawnd RC (9-13) insay wan klos lכp, bכt insay כda kכr kכmpleks dεm, transmεmbran pεptida dεm de intarap di kכntinyu fכ di LH1 we de rawnd am , Dεn de protεkt di kwinol/kwinכn difyushכn bitwin RC εn saytokrכm bc1 kכmpleks (11, 13-15). di pepul fכtotrofik plant Rhodopseudomonas (Rps.) na mכdel כganism we kin כndastand di enεji εn εlektrכn tכnfכs we de sכpכt fכtosεntesis. Di fɔs kristal strɔkchɔ fɔ Rps. di mכdel fכ di palustris RC-LH1 kכmpleks na RC, we de rawnd 15 hεterodimeric LH1 lכp dεm, we wan protin we dεn nכ no we dεn kכl “Protein W” (14) de intarap. Protein-W bin afta dat idεntify as RPA4402, we na wan uncharacterized 10.5kDa protin wit tri prεdikt transmembran hεlikεs (TMH) ( 16 ). wi prכpos fכ rinem di rpa4402 jin we de kכd protεin W to pufW fכ kכnsist wit di nכmenklachכ we dεn yuz fכ jin dεm we de kכd RC-L, M (pufL, pufM) εn LH1α, β (pufA, pufB) sכbyunit dεm. i intrestin fכ no se protin-W de כnli de insay bכt 10% pan RC-LH1, we de sho Rps. palustris de prodyuz tu difrεnt RC-LH1 kכmpleks dεm. Hia, wi ripot di ay-rεsכlushכn krayo-EM (cryo-EM) strכkchכ dεm fכ tu kכr kכmpleks, wan wit protin W εn 14 αβ hεterodaymεr, di כda wan we nכ gεt protin W εn wan klos 16 Hεterodaymεr LH1 lכp. wi strכkchכ de riprizent wan stεp chenj na di כndastandin fכ di RC-LH1 kכmpleks fכ Rps. palustris, biכs wi dכn analכz di homojεnik populeshכn fכ εvri vεryכnt εn wi gεt inof rεsכlushכn fכ klia wan asaynd εvri pεptida εn bכnd pigmεnt dεm εn rilayt lipid dεm εn kwinכn dεm. di kכmpεreshכn fכ dεn strכkchכ dεm ya sho se di tri TMH protin dεm-W we dεn nכ fכnshכn insay eni כda RC-LH1 kכmpleks so fa de jεnarεt wan kwinכn chεnal fכ aksεlεrayt di kwinכn/kwinכlכn εkshεnj. wan nכmba fכ kכnsyuv lipid εn kwinכn binding sayt dεm dεn dכn aydentify, εn wi dכn rivεl wan nyu kכnfכmeshכnal chenj afta di kכmbaynshכn fכ kwinכn εn RC, we kin fayn fכ fכtosistεm II (PSII) RC fכ כksijεn fכtotrofik כganism dεm. di tin dεm we wi fכnshכn de gi nyu insayt dεm fכ di kεnεtiks fכ kwinכn/kwinכlכn binding εn εkshεnj insay di RC-LH1 kכr kכmpleks fכ pכpul fכtotrofik baktri dεm.
fכ mek i izi fכ wan ditayl stכdi fכ di tu kכmpleks dεm we dεn fכnshכn na Rps. palustris, wi de aylכt εvri RC-LH1 bay bayokεmik mεtכd dεm. di protin W-deficient komplex (we dεn kכl am ΔpufW) bin purify frכm di strain we nכ gεt di pufW jin (16), εn na wan RC-LH1 kכmpleks nכmכ dεn kin prodyuz. di komplex we gεt protin W na wan strayn de mek am. di protin W fכ dis strayn de modify wit 10x His tεg na in C-tεrminus, so dat di protin W-kכntεn kכmpleks kin ifektivli kכmbayn wit mכst lכk protin W bay we i de imכbiliz mεtal. Di komplex de separet fayn fayn wan (16) Afiniti Kromatografi (IMAC).
as yu si na di pikchכ 1, di tu kכmpleks dεm gεt tri sכb-yunit RC (RC-L, RC-M εn RC-H) we wan LH1 εntena de rawnd. di 2.80-A strכkchכ fכ di kכmpleks we nכ gεt protin-W de sho 16 αβ hεterodaymεr dεm, we de fכm wan klos LH1 lכp we komplitli rawnd RC, we dεn kכl am afta dis RC-LH116 kכmpleks. di 2.65Å strכkchכ fכ di protin-W-kכntεn kכmpleks gεt 14-hεterodaymεr LH1 we protin-W intarכp, we dεn kכl am afta dis RC-LH114-W.
(A ɛn B) Surface ripreshɔn fɔ di kɔmpawnd. (C ɛn D) Bonded pigmɛnt dɛn we dɛn ɛksprɛs insay rod. (E εn F) di kכmpleks dεm we dεn obsεv frכm di saytoplasmik sεf gεt di pεptida dεm εn LH1 sεbyunit dεm we dεn rεprεsεnt insay katun, εn dεn nכmba kloksayz frכm di protin-W gap [kכnsist wit Rba nכmba. sphaeroides kɔmpleks (13)]. fכ LH1-α, di kכla fכ di protin sכbyunit na yכlכ; fכ LH1-β, di kכla fכ di protin sכbyunit na blu; fכ protin-W, di protin na rεd; fɔ RC-H, na sayn; fɔ RC-L, na Ɔrɛnj; fɔ RC-M, we na majenta. di kכfaktכr dεm de riprizent bay rod, grεn de riprizent BChl εn BPh a mכlikul dεm, pכpul de riprizent karotenoyd dεm, εn yכlכ de riprizent UQ10 mכlikul dεm. (G ɛn H) Magnifyed we fɔ si di protin-W gap na di ikwal rijyɔn fɔ RC-LH114-W kɔmpleks (G) ɛn RC-LH116 kɔmpleks (H). di kכfaktכr dεm de sho insay di fכm fכ spεs fכ fulכp, chεlat kwinכn de sho insay blu. di protin-W gap de hεlayt bay wan blu dεsh layn insay (G), εn di sכm sכm ol dεm usay kwinכn/kwinolכl de difכs na di LH116 ring dεn hεlayt bay wan blak dεsh layn insay (H).
Fig 1 (A εn B) sho di RC we dεn rawnd wit opin כ klos εryu dεm fכ LH1αβ hεterodaymεr dεm, εvri wan pan dεm de biεn tu BChl εn wan karotenoyd (Figure 1, C εn D). stכdi dεm we dεn bin dכn du bifo dis tεm dεn sho se Rps na di LH1 kכmpleks. insay di bayosεntetik path fכ spirulina xanthin, dεn spεshal wan dεm ya gεt miks populeshכn dεm fכ karotenoyd dεm ( 17 ). כltu, spiropyrroxanthin na di dominant karotenoyd εn in dεnsiti de satisfay. כl dis mek wi pik fכ mכdel spiroxanthin na כl di LH1 binding sayt dεm. di alfa εn beta polipεptida dεm na singl TMH dεm wit sכt mεmbran כta rijyכn dεm (Figure 1, A, B, E, εn F). pan tap we dεn nכ bin obsεv di dεnsiti fכ 17 rεsidεns na di C-tεrminus, di alfa pכlipεptida bin kכl frכm Met1 to Ala46 insay di tu kכmpleks dεm. β polypeptide bin ridyus frכm Gly4 to Tyr52 in RC-LH116, εn frכm Ser5 to Tyr52 in RC-LH114-W. No density fכ 3 כ 4 N-tεrminal כ 13 C-tεrminal rεsidεns dεm bin obsεv (Figure S1). Mas spεktrכmetri analisis fכ di miks RC-LH1 kכmpleks we dεn pripia frכm di wayl-tayp strayn sho se di mis rijyכn na di rizulεt fכ hεtεrכlכg klivεj fכ dεn pεpti dεm ya (Figure S1 εn S2). Di N-tεrminal fכmyleshכn כf α-Met1 bin כlso obsεv (f). di analisis sho se di α-pεptida kכnsis fכ rεsidεns fMet1 to Asp42/Ala46/Ala47/Ala50, εn di β-pεptida kכnsis fכ rεsidεns Ser2 to Ala53, we de in gud agrimεnt wit di lכw-tεmprachכ EM dεnsiti map.
kכdכnayshכn fכ α-His29 εn β-His36 de mek BChls fes to fes; εvri αβ hεterodaymεr de asembl wit in neba dεm fכ fכm wan opin lכp (RC-LH114-W) כ wan klos lכp (RC-LH116) rawnd di RC Di εksiton kכpl pigmεnt εry (Figure 1, C εn D). we yu kompare wit di 877 nm band fכ RC-LH114-W, di 880 nm absכpshכn rεd shift fכ RC-LH116 na 3 nm (Figure 2A). Bɔt di sɛkɔral dichroism spɛktrum na ɔlmost di sem (Figure 2B), we sho se pan ɔl we klia difrɛns de bitwin opin ɛn klos lɔp, di lokal ɛnvayrɔmɛnt fɔ BChls rili fiba. di absכpshכn rεdshift kin bi di rizulεt fכ ridyus tεmכl muvmεnt εn inkrεs stεbiliti pan di klos lכp (18, 19), di chenj in pigmεnt kכpl we di kכlos lכp kכz (20, 21), כ wan kכmbaynshכn fכ dεn tu ifekt dεm ya (11).
(A) Ultraviolet/visible/near-infrared absכpshכn spεktrum, di pik dεm we dεn mak wit dεn kכrεspכndεnt pigmεnt dεm εn nכmal to di BPh pik na 775 nm. (B) Sεkyulכr dichroism spεktrum nכmal to BChl absכrbεns na 805 nm. (C ɛn D) Sɛlɛkt ΔA spɛktra frɔm di tɛm-rɛzolt absɔpshɔn spɛktra fɔ RC-LH114-W kɔmpleks (C) ɛn RC-LH116 kɔmpleks (D). Fɔ bɛtɛ kɔmparabiliti, ɔl di spɛktra dɛn nɔmal to ∆A ɔf −A na 0.2 ps. (E) Di rεt fכ saytokrכm c2 כksidεshכn afta irεdyushכn in di prεsεns כf difrεn kכnsantreshכn כf UQ2 (luk fכg S8 fכ raw data). (F) insay sεl dεm we gro כnda lכw, mεdiכm כ hכy intensiti layt (10, 30 כ 300μMm-2 s-1, rispεktivli), di protin W εn RC-L sכbyunit dεm na di purifyed kכmpleks εn di separet mεmbran rεshכ. ditarmin di protin lεvεl bay SDS-polyacrylamide jel εlektrofכresis εn immunoassay (luk fכg S9 fכ raw data). Ditarmin di rεshכn rilitiv to di purifyed RC-LH114-W kכmpleks. di stoichiometrik rεshכn fכ RC-L to protin-W fכ di kכmpleks na 1:1.
di BChls na posishכn 1 na di difכm αβ14 lכp na RC-LH114-W (Figure 1, A, C, εn E) de klos to di RC praymar dכna (P) bay 6.8Å pas di ikwal BChls na RC-LH116 (Figure. 1, B, D, εn F, εn Fig S3); כltu, di transiεnt absכpshכn kεnεtiks fכ di tu kכmpleks dεm sho se fכ RC-LH114-W εn RC-LH116, di εksitayshכn εnεji tכnfכs tεm kכnstant frכm LH1 to RC na 40 ±4 εn 44±3 ps (Figure 2). , C ɛn D, Fig S4 ɛn Tebul S2). No signifyant difrɛns nɔ de bak pan ilɛktronik transfɔm insay RC (Figure S5 ɛn rilat suplimɛnt tɛks). wi sכspεkt se di klos kכrεspכndεns fכ di enεji tכnfכs tεm bitwin LH1 εn RC-P na biכs fכ di sεm kayn distans, angle εn pכtεnshal enεji fכ mכst BChl insay di tu LH1 lכp dεm. i lεk se fכ εksplכr di LH1 enεji patεn fכ rich di minim distans nכ de fast pas dayrεkt enεji tכnfכs frכm sabכptimal sayt dεm to RC. di opin-lכp LH1 lכp in RC-LH114-W kin כndago insignifikant tεmral mכshכn כnda lכw tεmprachכ kכndishכn fכ strכkchכral analisis, εn lכng αβ14 ring kכnfכmeshכn de na rum tεmprachכ frכm di pigmεntεshכn distans fכ βBChls na di posishכn fכ RC 1.
di RC-LH116 komplex gεt 32 BChls εn 16 karotenoyd dεm, εn in כvala arenjmεnt na di sem wit di wan we dεn gεt frכm Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 strayn ( PDB ID 7C9R) (12) εn grεn algae (Blc.viridis) (PDB ID 6ET5) (10) we de mek di bɔdi de. Afta alaynɛshɔn, na smɔl smɔl dɛvyayshɔn nɔmɔ dɛn si na di pozishɔn dɛn we αβ hɛtɛrodaym dɛn de, spɛshal wan 1-5, 15, ɛn 16 (Figure S6). di prεsεns fכ protin-W gεt signifyant impak pan di strכkchכ fכ LH1. in tri TMH dεm de kכnekt bay sכt lכp dεm, wit di N-tεrminal na di lumεn sayd fכ di kכmpleks εn di C-tεrminal na di saytoplasmik sayd (Figure 1A εn 3, A to D). di protin-W na big wan we de fכm haydrofobik (Figure 3B), εn TMH2 εn TMH3 de intarakt wit LH1αβ-14 fכ fכm wan transmembran sεf (Figure 3, B εn E to G). di intafeys de mεnli kכmכp fכ Phe, Leu εn Val rεsidεns dεm na di transmεmbran rijyכn. dis rεsidεns dεm de stak wit haydrofכbik amino asid dεm εn αβ-14 pigmεnt dεm. sכm polar rεsidεns dεm de kכntribyut bak to di intarakshכn, inklud di haydrojεn bכnd bitwin W-Thr68 εn β-Trp42 na di sεf fכ di kכmpleks kεviti (Figure 3, F εn G). pan di sεf fכ di saytoplasm, Gln34 de nia di kεto grup fכ αβ-14 karotenoyd dεm. apat frכm dat, di n-dodecyl β-d-maltoside (β-DDM) mכlikul bin rεsכlv, εn in haydrofכbik tel bin εkstendi to di intafεys bitwin protin-W εn αβ-14, εn di lipid tel kin de na di bכdi. wi notis bak se di C-tεrminal rεsכlushכn rijyכn dεm fכ protin W εn RCH de rili klos, bכt nכto insay di skop fכ fכm spεsifi k intarakshכn dεm (Figure 1, A εn E). כltu, intarakshכn kin de insay di כn rεsכlv C-tεrminal amino asid dεm fכ dεn tu protin dεm ya, we kin gi wan mεkanism fכ di rikrutmεnt fכ protin-W di tεm we di RC-LH114-W kכmpleks de asembli.
(A) Protein-W, we de fes di intafeys wit LH1αβ14 in katun fכm, gεt wan rod-shεp sayd chen (rεd), we dεn sho insay wan pat pan di ilektrostatik pכtεnshal dayagram (transparent grey sεf wit kכntor lεvεl we na 0.13). (B) Protein-W we dεn rεprεsεnt bay wan haydrofכbik kכla sεf. Polar ɛn chaj eria dɛn de sho insay sayn, di say dɛn we gɛt haydrofobik de sho wit wayt, ɛn di say dɛn we strɔng haydrofobik de sho wit ɔrɛnj. (C εn D) Protεin-W we dεn rεprεsεnt insay katun, in כryenteshכn na di sem lεk na (A) (C), εn i de rכt bay 180° (D). akɔdin to di pozishɔn we de na di sikwins, di rɛsɛdyu dɛn we dɛn kin difrɛns adopt wan renbo kɔlɔ skim, usay di N-tɛrminal na blu ɛn di C-tɛminal na rɛd. (E) Protein-W in di sem view as in (A), εn di rεsidεns dεm na di intafεys fכ protin-W:LH1 dεn riprizent bay rod dεm wit mak dεm we dεn atak. (F) Protein-W de rota 90° rilitiv to (E) ɛn LH1αβ14 insay di katun ripreshɔn, ɛn rilitiv to di intafɛs rɛsɛdyu na di bar ripreshɔn. di ovahangin rεsidεns frכm di beta polipεptida dεn de lεbul. di kכfaktכr dεn sho am as bar we de mach di kכla na fכg 1, di dεkכmpכz β-DDM dεn sho am wit grey, εn di כksijεn dεn sho am wit rεd. (G) di vεyushכn na (F) de rכt 180°, wit di prominεnt rεsidεns dεm fכ di lεbul alfa pכlipεptida.
protεin-W de riples wan αβ hεterodaymεr (di 15th na fכg 1F), we de mek di lכp kכloz εn tilt di fכs tri αβ hεterodaymεr dεm. I bin obsεv se di maksimal inklinεshכn angle fכ di fכs αβ-1 hεterodaymεr rilitiv to di film nכmal na 25° to 29° (Figכ 1, A εn E), we bin fכm bay di 2° to 8° inklinεshכn כf αβ-1 in RC A shap kכntrast-LH116 (Figכ 1, B εn F). di sεkכn εn tכd hεterodaymεr dεm de inklin na 12° to 22° εn 5° to 10°, rispεktivli. fכ di stεrik hindrεns fכ RC, di tilt fכ αβ-1 nכ inklud di sεkכn pe fכ αβ (we kכrεspכnd to di 16th αβ na fכg 1F), so dat de fכm wan klia gap insay di LH1 ring (Figכ 1, A εn E) . biכs fכ di lכk fכ tu αβ hεterodaymεr dεm, we kכmpan wit di lכs fכ BChl εn tu karotenoyd dεm, nכn pan di karotenoyd dεm de biεn di twist αβ-1 sabyunit, we de rizulta in wan LH114-W ring we gεt 13 karotenoyd dεm Vegetarian εn 28 BChls. di lokal rεsכlushכn εstimat dεm fכ di tu kכmpleks dεm na di αβ1 to 7 rijyכn dεm lכw pas di wan dεm fכ di rεst כf di LH1 lכp, we kin rεflek di inhεrent plastisiti fכ di LH1 sεbyunit adjasent to di RC QB sayt (Figure 4) .
di pikchכ dεm fכ RC-LH114-W (A εn B) εn RC-LH116 (C εn D) dεn sho frכm di sem tכp vεyu/sayd vεyu (A εn B) (A εn C) εn kaviti sεf na Fig. 1. (B εn D). Di ki dɛn we gɛt kɔlɔ de sho na di rayt say.
di onli oda kכntribyushכn kכr kכmpleks wit stoichiometrik rεshכn we na 1:14 na di Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). כltu, protin W εn PufX nכ gεt nכ klia homכli, εn dεn gεt sכm impak pan dεn rispεktiv LH1 strכkchכ dεm. PufX na wan TMH wit wan N-tεrminal saytoplasmik domεn we de intarakt wit di saytoplasmik sayd fכ di RC-H sεbyunit (13) na wan posishכn we kכrεspכnd to Rps. palustris LH116αβ-16 we de na di bɔdi. PufX de mek wan chanεl fכ di kwinכn/kwinכlכn εkshεnj bitwin RC-LH1 εn di saytokrכm bcl kכmpleks εn i de insay כl di Rba. sphaeroides kor komplex (13). Although di monomer-monomer intafeys de na Rba. di sphaeroides RC-LH1-PufX dimer de na di binding posishכn fכ protin W insay RC-LH114-W, εn di gap we PufX εn protin-W indyuz de na wan ikwal posishכn (Figure S7A). di gap insay RC-LH114-W de alaynd bak wit di haypothεtik kvinכn chεnal (8) fכ Pseudomonas rosea LH1, we dεn fכm bay pεptida dεm we nכ rilet to protin W כ PufX (Figure S7B). Apat frɔm dat, di kinɔn chanɛl we de na Blc. di εmerald grεn LH1 we fכm bay we dεn kכl wan γ sεbyunit (7) de na wan sεm kayn posishכn (Figure S7C). pan tap we difrεn protin dεm de mεdiet am, di apiyans fכ dεn kwinכn/kwinol chεnal dεm ya na kכmכn posishכn na di RC-LH1 kכmpleks lεk se na egzampl fכ kכnvεrjεnt εvכlyushכn, we sho se di gap we protin W de mek kin akt lεk kwinכn chεnal.
di gap na di LH114-W lכp de alaw fכ fכmeshכn fכ wan kכntinyu mεmbran rijyכn bitwin di intanal spεs fכ di RC-LH114-W kכmpleks εn di bכku mεmbran (Figure 1G), pas fכ kכnekt di tu domεn dεm tru wan protin por lεk insay protin dεm. Di RC-LH116 komplex na di sem wit wan klos Tch. Komplex we tan lɛk nidul (22) (Figure 1H). biכs di difyushכn fכ kwinכn tru di mεmbran de fast pas di difεsכn tru di sכm protin chεnal, di opin LH114-W lכp kin alaw fכ RC tכnכv fast pas di kכlos LH116 lכp, εn di difyushכn fכ kwinכn insay di RC kin bi mכr rεstrikt. fכ tεst if protin W de afekt di kכnvכshכn fכ kwinכn tru RC, wi du wan saytokrכm כksidεshכn asεy pan wan sכm kכnsantreshכn fכ ubikinכn 2 (UQ2) (wan analכg fכ nεchכral UQ10 wit sכt isoprene tel) (Figure 2E). pan tap we di prεsεns fכ chεlat kwinכn de hεndεr di akכrd ditarminεshכn fכ di apεrεnt Maykεlis kכnstant (RC-LH114-W εn RC-LH116 na suitable fכ 0.2±0.1μM εn 0.5±0.2μM, rispεktivli), di maksimal rεt fכ RC-LH114-W ( 4.6±0.2 e-RC-1 s-1) na 28±5% big pas RC-LH116 (3.6±0.1 e-RC-1 s-1).
wi fכs εstimat se protin-W de insay bכt 10% pan di kכr kכmpleks ( 16 ); na ya, di okyupashכn rεt fכ lכw-layt, mεdiכm-layt, εn ay-layt gכt sεl dεm na 15±0.6%, 11±1% εn 0.9±0.5, rispεktivli % (Figure 2F). Kwantiti kכmpεreshכn כf mas spεktrכmetri sho se di adishכn כf histidine tag nכ ridyus di rilitiv abundans כf protin-W kכmpεr to wayl-tayp strayn (P = 0.59), so dεn lεvεl ya nכto artifakt כf modifyed protin-W (Figure S10). כltu, dis lכw okyupashכn fכ protin-W insay di RC-LH1 kכmpleks kin alaw sכm RC dεm fכ flip pan aksεlεrayt rεt, we de mitigate di slo kwinכn/kwinכlכn εkshεnj insay di RC-LH116 kכmpleks. wi notis se di ay layt okyupashכn rεt nכ kכnsist wit di rεsεnt transkriptכmiks data, we sho se pufW jin εksprεshכn de inkrεs כnda strכng layt (Figure S11) (23). di difrεns bitwin pufW transkripshכn εn protin-W inkכrporεshכn insay di RC-LH1 kכmpleks de kכnfyus εn i kin rεflek di kכmpleks rεguleshכn fכ di protin.
insay RC-LH114-W, 6 kadiolipin (CDL), 7 fכsfatidylkolin (POPC), 1 fכsfatidylglisεrol (POPG) εn 29 β-DDM mכlikul dεm dεn alכk εn mכdel insay am 6 CDL dεm, 24 POPC dεm, 2 POPG dεm εn 12 βDDM dεm. RC-LH116 (Figure 5, A ɛn B). insay dεn tu strכkchכ dεm ya, CDL de kכlכsכl na di saytoplasmik sayd na di kכmpleks, we POPC, POPG εn β-DDM de mכst na di luminal sayd. Tu lipid ɛn ditarjen mɔlyul dɛn bin aysol insay di αβ-1 to αβ-6 rijyɔn na di RC-LH114-W kɔmpleks (Figure 5A), ɛn fayv bin aysol insay di ikwal rijyɔn na RC-LH116 (Figure 5B ). mכr lipid dεm bin fכn na di כda say na di kכmpleks, mεnli CDL, we dεn akumulet bitwin RC εn αβ-7 to αβ-13 (Figure 5, A εn B). כda strכkchכral rεsכlv lipid dεm εn ditarjεnt dεm de ausayd di LH1 ring, εn wel-rεsכlv asil chen dεm de εksεnd bitwin LH1 sεbyunit dεm, we dεn tεntivli dεsignet as β-DDM insay RC-LH114-W, εn dεfεn dεm as β-DDM insay RC A miksכp כf β-DDM εn POPC-LH116. di sεm kayn posishכn dεm fכ di chεlat lipid dεm εn ditarjen dεm na wi strכkchכ de sho se dεn na fysiolojikal rilevεnt binding sayt dεm (Figure S12A). di posishכn dεm fכ ikwal mכlikul dεm na Tch dεn tu gεt gud kכnsistεns. Jɛntil ɛn Trv. Strain 970 RC-LH1s (Figure S12, B to E) (9, 12) εn di haydrojen-bכnding rεsidεns dεm fכ di lipid hεd grup sho fεy gud kכnsyuvεshכn in di sikεns alaynsmεnt (Figure S13), we sho se Konsavכv CDL we de biεn to RC (24), dεn sayt dεm ya kin kכnsyuv insay di RC-LH1 kכmpleks.
. UQ we bכnd to RC QA εn QB sayt dεm na yכlכ, we isol UQ na blu. (C εn D) Di sem we aw pipul dεn de si (A) εn (B), wit di lipid dεm we dεn nכ de. (E to G) Enlarged view of Q1(E), Q2(F) and Q3(G) frɔm RC-LH116, wit sayd chen dɛn we de inflɔws dɛnsɛf. Dɛn sho di haydrojɛn bɔnd dɛn as blak dɛsh layn dɛn.
insay RC-LH116, כl tu di RC QA εn QB UQ, we de tek pat pan εlektrכn tכnfכs insay di chaj separeshכn prכsεs, de dכn kכmכpכz insay dεn binding sayt dεm. Bɔt insay RC-LH114-W, dɛn nɔ dɔn sɔlv QB kwinɔn ɛn dɛn go tɔk bɔt am ditayli dɔŋ ya. apat frכm QA εn QB kwinכn dεm, tu chεlat UQ mכlikul dεm (we de bitwin di RC εn LH1 ring dεm) dεn alɔkat insay di RC-LH114-W strכkchכ akɔdin to dεn hεd grup dεm we dεn rεsכlv fayn fayn wan (we de insay Q1 εn Q2, rispεktivli). spes). Fig 5C) we de na di pikchɔ. Tu isoprene yunit dεm dεn asaynd to Q1, εn di dεnsiti map de rεsכlv di komplit 10 isoprene tel dεm fכ Q2. insay di strכkchכ fכ RC-LH116, tri chεlat UQ10 mכlikul dεm (Q1 to Q3, Figure 5D) bin rεsכlv, εn כl mכlikul dεm gεt klia dεnsiti tru di tel (Figure 5, D to G). insay di tu strכkchכ dεm, di posishכn dεm fכ di kwinכn hεd grup dεm fכ Q1 εn Q2 gεt εksεlεnt kכnsistεns (Figכ S12F), εn dεn nכmכ de intarakt wit RC. Q1 de na di ɛntrɛ na di W gap na RC-LH114-W (Figure 1G ɛn 5, C, D ɛn E), ɛn Q2 de nia di QB binding sayt (Figure 5, C, D) ɛn F). di kכnsyuv L-Trp143 εn L-Trp269 rεsidεns dεm de vεri klos to Q1 εn Q2 εn de gi pכtεnshal π-stak intarakshכn (Figכ 5, E εn F, εn Fig S12). L-Gln88, 3.0 Å frכm di distal כksijεn fכ Q1, de gi strכng haydrojεn bכnd (Figure 5E); dis rεsidεns de kכnsyuv insay כl di RC dεm εksεpt di rilayshכn we de fa pas כl (Figure S13). L-Ser91 na kכnsyuvli sכbstityuyt fכ Thr in mכst כda RC dεm (Figure S13), na 3.8 Angstroms frכm di mεtyl כksijεn fכ Q1, εn i kin gi wik haydrojεn bכnd dεm (Figure 5E). i tan lεk se Q3 nכ gεt spεsifi k intarakshכn, bכt i de na di haydrofכbik rijyכn bitwin di RC-M sכbyunit εn di LH1-α sכbyunit 5 to 6 (Figure 5, D εn G). Q1, Q2 εn Q3 כ nia chelated quinones dεn dכn rεsכlv bak insay Tch. Jɛntil, Trv. Strain 970 ɛn Blc. di iris strכkchכ (9, 10, 12) de pכynt to wan kכnsyuv כksijεn kvinכn binding sayt insay di RC-LH1 kכmpleks (Figure S12G). Di fayv dikɔmpɔz UQ dɛn na RC-LH116 de in gud agrimɛnt wit di 5.8±0.7 pan ɛni kɔmpleks we dɛn disayd bay ay pafɔmɛnshɔn likwid kromatografi (HPLC), we di tri dikɔmpɔz UQ dɛn na RC-LH114-W smɔl pas Di mɛzhɔ valyu we na 6.2±0.3 (Fig. S14) sho se UQ mɔlyul dɛn de we nɔ sɔlv insay di strɔkchɔ.
di pseudo-symmetric L εn M polipεptida dεm εvri wan gεt fayv TMH dεm εn fכm wan hεterodaymεr we de kכmbayn wan BChl dimer, tu BChl monomer, tu bakteriophage (BPh) monomer, εn wan nכn- Heme ayron εn wan כ tu UQ10 mכlikul dεm. tru di prεsεns fכ haydrojεn bכnd dεm na di tεrminal kεtכn grup εn in akyumyuleshכn we dεn no insay Rps, karotenoyd dεm de inkכrporεt insay di M-sכbyunit, we dεn kכl cis-3,4-dehydroorhodopin. Di spɛshal wan dɛn (25). di כta mεmbran domεn fכ RC-H de ankכr to di mεmbran bay wan singl TMH. di ovala RC strכkchכ na di sem wit di tri sכbyunit RC fכ riletid spεshal (lεk Rba). sfɛroyd dɛn (PDB ID: 3I4D). di makrosaykl dεm fכ BChl εn BPh, di karotenoyd bakbon εn nכn-hεm ayכn de כvalap insay di rεsכlushכn rεnj fכ dεn strכkchכ dεm ya, lεk aw di UQ10 hεd grup na di QA sayt εn di QB kwinכn na RC-LH116 (Figure S15).
di avεlayblεti fכ tu RC strכkchכ dεm wit difrεnt QB sayt okyupashכn rεt de gi nyu chans fכ egzamin di kכnsistεnt kכnfכmeshכnal chenj dεm we de akkompani QB kwinכn binding. insay di RC-LH116 komplex, QB kwinכn de na di ful bכnd “proksimal” posishכn ( 26 ), bכt di separeshכn fכ RC-LH114-W nכ gεt QB kwinכn. nכ QB kwinכn de insay RC-LH114-W, we de sכprayz biכs di kכmpleks de aktv, mכr pas di RC-LH116 kכmpleks wit strכkchכral rεsכlv QB kwinכn. pan tap we di tu LH1 ring dεm de chelet bכt siks kwinכn dεm, fayv dεn strכkchכral rεsכlv insay di kכlos RC-LH116 ring, we na tri nכmכ strכkchכral limited in di opin RC-LH114-W ring . dis inkrεs strכkchכral dizכrd kin rεflεkt di fast riplesmεnt fכ RC-LH114-W QB sayt dεm, fasta kvinכn kεnεtiks insay di kכmpleks, εn inkrεs liklihood fכ kכros di LH1 lכp. wi se di lכk fכ UQ na di RC QB sayt fכ RC-LH114-W kin bi di rizulεt fכ wan mכr komplεks εn mכr aktv kכmpleks, εn di QB sayt fכ RC-LH114-W dεn bin froz wantεm wantεm insay UQ tכnכv Di spεsifi k stej (dεn dכn klos di εntrεs to di QB sayt) de rεflek di kכnfכmeshכn fכ dis aktvכti.
if QB nכ de, di akkompani rotashכn fכ L-Phe217 to wan posishכn we inkompatibl wit UQ10 binding, biכs i go mek wan spatial kכlishכn wit di fכs isoprene yunit fכ di tel (Figure 6A). apat frכm dat, di klia men kכnfכmeshכnal chenj dεm de klia, spεshal wan di hεliks de (sכt hεliks na di lכp bitwin TMH D εn E) usay L-Phe217 de shift to di QB binding pכket εn di roteshכn fכ L-Tyr223 (Figure 6A ) Fכ brok di haydrojεn bכnd wit di M-Asp45 fכm εn klos di εntrεs fכ di QB binding sayt (Figure 6B) we de na di wɔl. Hεliks de pivot na in bays, di Cα fכ L-Ser209 de shift bay 0.33Å, we di L-Val221Cα de shift bay 3.52Å. nכ chenj dεm de we dεn kin si pan TMH D εn E, we dεn kin supεrimכz insay di tu strכkchכ dεm (Figure 6A). as wi no, dis na di fכs strכkchכ na di nεchכral RC we de kכloz di QB sayt. we yu kכmpεr wit di komplit (QB-bound) strכkchכ sho se bifo di kwinכn dכn rεdכks, kכnfכmeshכnal chenj nid fכ mek i go insay di kwinכn. L-Phe217 de rotate fכ fכm π-stacking intarakshכn wit di quinone hεd grup, εn di hεliks de shift autsay, we de alaw di skel fכ L-Gly222 εn di sayd chen fכ L-Tyr223 fכ fכm wan haydrojεn bכnd nεtwכk wit wan stebul haydrojεn bכnd strכkchכ (Figure 6, A εn C).
(A) Ovalap katun we gɛt ɔlogram (L chen, ɔrɛnj/M chen, majenta) ɛn apo (grey) strɔkchɔ, we di ki rɛsɛdyu dɛn de sho insay di fɔm fɔ wan ripreshɔn we tan lɛk rod. UQ10 na wan yɔlɔ bar we dɛn ripresent. Di dɔt layn de sho di haydrojɛn bɔnd dɛn we dɔn fɔm insay di wan ol strɔkchɔ. (B εn C) di sכfayz rεprεsεntεshכn fכ di apolipoprotein εn di ol ring strכkchכ, we de hεlayt di sayd chen כksijεn fכ L-Phe217 in blu εn L-Tyr223 in rεd, rispεktivli. Di L sabyunit na ɔrɛnj; di M εn H sכbyunit dεm nכ gεt kכla. (D ɛn E) Apolipoprotein (D) ɛn ɔl (E) RC QB sayt dɛn [kɔlɔ bay (A) rispɛktvɔli] ɛn Thermophilus thermophilus PSII (grin, blu wit plastic kwinɔn; PDB ID: 3WU2) Align (58).
we wi nכ bin εkspεkt, pan כl we sεvεra strכkchכ dεm fכ QB-dεficient RC dεm we nכ gεt LH1 de, di kכnfכmeshכnal chenj dεm we dεn obsεv insay dis stכdi nכ bin ripot bifo. Dis inklud di QB diplεshכn strכkchכ frכm Blc. viridis (PDB ID: 3PRC) (27), Tch., ɛn ɔda pipul dɛn. tepidum (PDB ID: 1EYS) (28) ɛn Rba. sphaeroides (PDB ID: 1OGV) (29), ol dεm na kכlכsכl di sem wit dεn כvala QB strכkchכ. Klos inspekshכn fכ 3PRC sho se LDAO (Lauryl Dimethyl Amine Oxide) ditarjent mכlikul dεm de biεn na di εntrεs fכ di QB posishכn, we kin mek dεn nכ riarenjmεnt insay wan kכlos kכnfכmeshכn. Pan ɔl we LDAO nɔ de rɔtin na di sem pozishɔn na 1EYS ɔ 1OGV, dɛn kin pripia dɛn RC ya bay we dɛn de yuz di sem ditajɛnt ɛn dat mek dɛn kin mek di sem ifɛkt. Di kristal strɔkchɔ na Rba. Sphaeroides RC we kכ-kristalayz wit saytokrכm c2 (PDB ID: 1L9B) dεn tu lεk se i gεt wan klos QB sayt. כltu, in dis kes, di N-tεrminal rijyכn fכ di RC-M pכlipεptida (we de intarakt wit di QB binding sayt tru di H bכnd fכ di Tyr rεsidyu pan di Q hεliks) de adopt wan כnnatural kכnfכmeshכn, εn di QB kכnfכmeshכnal chenj nכ de fכs εksplכr (30 ). wetin de mek wi fil fayn na dat wi nכ si dis kayn difכmeshכn fכ di M polipεptida insay di RC-LH114-W strכkchכ, we kכlכsכl di sem wit di N-tεrminal rijyכn fכ RC-LH116 RC. i fכ no bak se afta di eradikeshכn fכ di ditarjen-bεys LH1 εntena, di apolipoprotein RC dεm na di PDB bin rεsכlv, we εliminet di intanεt kwinכn pul dεm εn lipid dεm na di gap bitwin di RC εn di insay sεf fכ di LH1 ring we de rawnd (31, 32). RC de stil fכnshכnal biכs i de rεtεn כl di kכfaktכr dεm, εksεpt fכ di kכmכpכzabl QB kwinכn, we nכ stebul εn i kin lכs bכku tεm di tεm we di prεparashכn prכsεs de (33). apat frכm dat, dεn no se di rimobul fכ LH1 εn nεchכral saykli lipid dεm frכm RC kin gεt impak pan fכnshכn dεm, lεk di sכt layfspan fכ di chaj-sεparayt P+QB-stet (31, 34, 35). So, wi spεkul se di egzistens fכ di lokal LH1 ring we de rawnd di RC kin mεnten di “klos” QB sayt, so dat i kin kip di lokal envayroment nia di QB.
pan tap we apolipoprotein (witout QB quinone) εn di komplit strכkchכ de riprizent כnli tu snapshot fכ di tכnכv fכ di QB sayt, pas wan siriכs ivent, indikεshכn dεm de se di binding kin gεt fכ prεvεnt ribaynd bay haydrokwinכn Fכ inhibit sabstεrayt inhibishכn. di intarakshכn bitwin quinolol εn quinone nia di QB sayt fכ apolipoprotein kin difrεnt, we de mek RC rijek am. fכ lכng tεm dεn bin prכpos se kכnfכmeshכnal chenj dεm de ple rol fכ di binding εn rεdukshכn fכ di kwinכn dεm. di abiliti fכ frozεn RC dεm fכ ridyus kwinכn afta dak adapteshכn de impεr (36); X-ray kristalografi sho se dis damej de bi biכs fכ QB quinones we de trכp insay wan “distal” kכnfכmeshכn we de bכt 4.5 Å frכm di aktv proksimal posishכn (26) , 37). wi sכgεst se dis distal binding kכnfכmeshכn na sεnsshכt fכ di intamεdiεt stet bitwin apolipoprotein εn di ful ring strכkchכ, we de fכlכ di initial intarakshכn wit kwinכn εn di opin fכ di QB sayt.
di tayp II RC we dεn fכnshכn na di PSII kכmpleks fכ sכm fכtotrofik baktri dεm εn saynobakteria, algi εn plant dεm gεt strכkchכral εn fכnshכnal kכnsyuvεshכn (38). di strכkchכral alaynsmεnt we dεn sho na fכg 6 (D εn E) de εnfaz di similitu bitwin PSII RC dεm εn di QB sayt fכ di baktriyal RC kכmpleks. dis kכmpεreshכn dεn bin lכng tεm bi wan mכdel fכ stכdi di klos rilayt sistεm dεm fכ kwinכn binding εn rεdukshכn. di prεviכs pכblisheshכn dεm bin sכgεst se kכnfכmeshכnal chenj dεm de kכmכn wit PSII rεdukshכn fכ kwinכn dεm ( 39 , 40 ). fכ dat, we wi tink bכt di evolushכnal kכnsyuvεshכn fכ RC, dis binding mεkanism we dεn nכ bin obsεv bifo kin aplay bak to di QB sayt fכ PSII RC insay כksijεn fכtotrofik plant dεm.
Rps ΔpufW (unlabeled pufW dileshכn) εn PufW-His (C-tεrminal 10x His-tag protin-W we dεn εksprεs frכm di nεchכral pufW lכkus) strayn dεm. palustris CGA009 bin dεskrεb in wi fכs wok (16). Dis strayn dεm εn di isogenic wayl-tayp parent bin rεkכv frכm di friza bay we dεn strik sכm nכmba כf sεl dεm pan PYE (εvri wan 5 g lita -1) (dεn stכr insay LB na -80 °C, we kכntεn 50% ( w/v) glycerol) protin, yist εkstrakt εn succinate) agar [1.5% (w/v)] plet. Di plet bin inkubayt ɔvnayt insay dak na rum tɛmpracha ɔnda anaerobik kɔndishɔn, ɛn afta dat dɛn layt am wit wayt layt (~50 μmolm-2 s-1) we OSRAM 116-W halojen bɔl dɛn (RS Components, UK) gi fɔ 3 to 5 dez te wan singl kɔloni apia. A singl kכloni bin yuz fכ inokulεt 10 ml M22+ mεdiכm (41) we dεn supliment wit 0.1% (w/v) kasamino asid (we dεn kכl am M22). Di kɔlchɔ bin gro ɔnda lɔw ɔksijɛn kɔndishɔn na dak na 34°C wit shek pan 180 rpm fɔ 48 awa, ɛn afta dat dɛn inokulet 70 ml pan di kɔlchɔ ɔnda di sem kɔndishɔn fɔ 24 awa. wan sεmi-aerobik kכltכr wit volyum we na 1 ml dεn yuz fכ inokulεt 30 ml M22 mεdiכm insay 30 ml yunivεsal skru-tכp transparent glas bכtul εn irεdyet wit agitεshכn (~50μmolm-2 s-1) fכ 48 awa bay stεril magnεtik fכs Stirring rod. afta dat 30 ml pan di kכltכr bin inokul wit bכt 1 lita kכlt כnda di sem kכndishכn, we dεn yuz fכ inokulεt bכt 9 lita kכlt we i layt na ~200 μmolm-2 s-1 fכ 72 awa. Di sel dεm bin avεst bay sεntrifugayshכn na 7132 RCF fכ 30 minit, rεsuspεnd insay ~10 ml 20 mM tris-HCl (pH 8.0), εn stכr na -20°C te i nid.
afta yu tכw, ad sכm kristכl dεm fכ dioxyribonuklease I (Merck, UK), lysozyme (Merck, UK) εn tu Roche holoenzyme protease inhibitor tablets (Merck, UK) to di rεsuspεnd sεl dεm. Insay wan 20,000 psi Frɛnch prɛshɔn sɛl (Aminco, Amɛrika), dɛn bin ambɔg di sɛl dɛn 8 to 12 tɛm. Afta we dεn pul di sεl dεm we nכ brok εn di dεbris we nכ de sכlv bay sεntrifugayshכn na 18,500 RCF fכ 15 minit na 4°C, di mεmbran bin prεsipitεt frכm di pigmεnt layzεt bay sεntrifugayshכn na 113,000 RCF fכ 2 awa na 43,000°C. trowe di sכlubul frakshכn εn rεsuspεnd di kכla mεmbran insay 100 to 200 ml 20 mM tris-HCl (pH 8.0) εn homogenize te nכ agregεt dεm we yu kin si. Di suspεnd mεmbran bin inkכba insay 20 mM tris-HCl (pH 8.0) (Anatrace, USA) we kכntεn 2% (w/v) β-DDM fכ 1 awa insay dak na 4°C wit jεntεl stirring. Dɔn sɛntrifyuj na 70°C fɔ sɔlv 150,000 RCF na 4°C fɔ 1 awa fɔ pul di rɛsɛdyuɛl insɔlubul dɛn.
Di solubilizing membran frɔm di ΔpufW strain bin aplay to wan 50 ml DEAE Sepharose ayɔn ɛkshɛnj kɔlɔm wit tri kɔlɔm volyum (CV) fɔ binding bafa [20 mM tris-HCl (pH 8.0) we gɛt 0.03% (w / v) β-DDM]. Wash di kכlכm wit tu CV binding bכfa dεm, εn afta dat was di kכlכm wit tu binding bכfa dεm we gεt 50 mM NaCl. Di RC-LH116 komplex bin eluted wit wan linya gradient we na 150 to 300 mM NaCl (insay binding bafa) pan 1.75 CV, ɛn di rεmεnεnt binding kכmpleks bin eluted wit wan binding bafa we kכntεn 300 mM NaCl pan 0.5 CV. Kכlekt di absכpshכn spεktrum bitwin 250 εn 1000 nm, kip di frakshכn wit absכpshכn rεshכ (A880/A280) we big pas 1 na 880 to 280 nm, dilכt am tu tεm insay di binding bכfa, εn yuz di sem prosidכs bak pan di DEAE kכlכm On purification. Dilute di frakshɔn dɛn wit A880/A280 ratios we ay pas 1.7 ɛn A880/A805 ratios we ay pas 3.0, du di tɔd rawnd fɔ ayɔn ɛkshɛnj, ɛn kip frakshɔn dɛn wit A880/A280 ratios we ay pas 2.2 ɛn A880/A805 ratios we ay pas 5.0. Di patli purifyed komplex bin kɔnsɛntret to ~2 ml insay wan Amicon 100,000 mɔlikul wet kɔt-ɔf (MWCO) sɛntrifugal filta (Merck, UK), ɛn lod pan wan Superdex 200 16/600 saiz ɛksklɔzhɔn kɔlɔm (GE Healthcare, US) we gɛt 200 mM NaCl bafa, ɛn afta dat dɛn eluted insay di sem bafa na 1.5 CV. Kɔlkt di absɔpshɔn spɛktra fɔ di saiz ɛksklɔzhɔn frakshɔn, ɛn kɔnsɛntret di absɔpshɔn spɛktra wit A880/A280 rɛsɛshɔn ova 2.4 ɛn A880/A805 rɛsɛshɔn ova 5.8 to 100 A880, ɛn yuz dɛn wantɛm wantɛm fɔ krayo-TEM grid pripiamɛnt ɔ stɔrɔj ​​Kip na -80°C te nid.
Di solubilizing membran frɔm PufW-His strain bin aplay to wan 20 ml HisPrep FF Ni-NTA Sepharose kɔlɔm (20 mM tris-HCl (pH 8.0) we gɛt 200 mM NaCl ɛn 0.03% (w/w)) insay IMAC bafa ( GE Healthcare). v) β-DDM] we de mek di bɔdi. Di kɔlɔm bin was wit fayv CV dɛn we gɛt IMAC bafa, ɛn afta dat wit fayv CV dɛn we gɛt IMAC bafa we gɛt 10 mM histidin. Di kor komplex bin eluted frכm di kכlכm wit fayv IMAC bכfa dεm we kכntεn 100 mM histidine. di frakshכn we gεt di RC-LH114-W komplεks dεn kכnsantreyt to ~10 ml insay wan stεr tank we gεt wan Amicon 100,000 MWCO filta (Merck, UK), dεn dεl am 20 tεm wit binding bכfa, εn afta dat dεn ad am to 25 ml Insay di DEAE Sepharose kכlכm, dεn yuz fכ CV dεm we bכnd to di bכfa bifo tεm. Wash di kɔlɔm wit 4 CV binding bafa, dɔn elute di kɔmpleks pan et CV dɛn pan wan linya gradient we na 0 to 100 mM NaCl (insay binding bafa), ɛn di ɔda 4 CV dɛn we gɛt 100 mM binding bafa Di rɛsɛdyuɛl kɔmpleks dɛn we dɛn eluyt pan di sɔdyɔm klorayd we dɛn jɔyn wit di A880/A280 rishɔ we ay pas 2.4 ɛn di A880/A805 ratio we ay pas 4.6 frakshɔn dɛn bin kɔnsɛntret to ~2 ml insay wan Amicon 100,000 MWCO sɛntrifugal filta, ɛn ful-ɔp wit 1.5 CV IMAC bifo tɛm Buffer equilibrated Superdex 200 16/600 saiz ɛksklɔzhɔn kɔlɔm, ɛn afta dat dɛn eluted insay di sem bafa ova 1.5 CV. Kɔlkt di absɔpshɔn spɛktra dɛn fɔ di saiz-ɛksklushɔn frakshɔn dɛn ɛn kɔnsɛntret di absɔpshɔn spɛktra dɛn wit A880/A280 rɛsɛshɔn dɛn ova 2.1 ɛn A880/A805 rɛsɛshɔn dɛn ova 4.6 to 100 A880, we dɛn kin yuz wantɛm wantɛm fɔ frɔzɛn TEM grid pripiamɛnt ɔ dɛn kin kip na -80°C te nid.
Wan Leica EM GP imɛshɔn friza bin yuz fɔ pripia lɔw tɛmpracha TEM grid dɛn. Di komplex bin diluted in IMAC bafa to A880 of 50, ɛn afta dat 5μl bin lod pan wan nyu glɔ-dischaj QUANTIFOIL 1.2/1.3 kabɔn-kɔt kɔpa mɛsh (Agar Scientific, UK). Inkubayt di grid na 20°C ɛn 60% rilitiv humiditi fɔ 30 s, dɔn blot am dray fɔ 3 s, ɛn afta dat, kwɛnch am insay likwid ɛtɛn na -176°C.
Di data fɔ di RC-LH114-W kɔmpleks bin rikodɔ pan di eBIC (Ilɛktronik Bayoimajin Sɛnta) (British Daymɔnd Layt Sɔs) wit wan Titan Krios maykroskɔp, we de wok pan aksilarayshɔn vɔltɛm we na 300kV, wit wan nominal magnifyeshɔn we na 130,000× ɛn wan ɛnaji we na- Pik wan gap we na 20 eV. Wan Gatan 968 GIF Kwantum wit K2 pik ditektɔ bin yuz fɔ rikodɔ imej dɛn insay kɔntin mɔd fɔ gɛda data. di kalibrayt piksεl saiz na 1.048Å, εn di dכz rεt na 3.83 e-Å-2s-1. I gɛda di fim insay 11 sɛkɔn ɛn sheb am to 40 pat dɛn. Yuz di say we dɛn kɔt di kabɔn fɔ rifokus di maykroskɔp, dɔn gɛda tri fim dɛn fɔ ɛni ol. In totכl, 3130 fim dεm bin kכlekt, wit difכkus valyu bitwin -1 εn -3μm.
Di data fɔ di RC-LH116 kɔmpleks bin kɔlɛkt yuz di sem maykroskɔp na di Asterbury Biostructure Laboratory (University of Leeds, UK). Di data bin kכlekt in kכunt mכd wit magnifyushכn 130 k, εn di piksεl saiz bin kalibrayt to 1.065 Å wit dכz 4.6 e-Å-2s-1. Dɛn bin rɛkɔd di fim insay 12 sɛkɔn ɛn dɛn sheb am to 48 pat dɛn. In totכl, 3359 film dεm bin kכlekt, wit difכkus valyu bitwin -1 εn -3μm.
Ɔl di data prɔsesin de du insay di Relion 3.0 paip layn (42). Yuz Motioncorr 2 (43) fɔ kɔrɛkt di bim muvmɛnt bay we yu de weit di doz, ɛn afta dat yuz CTFFIND 4.1 (44) fɔ no di CTF (kɔntrast transfɔm fɛnshɔn) paramita. Tipik fכtomaykrograf dεm afta dεn initial prכsεsin stej dεm ya dεn sho na fכg 2. S16. Di ɔtomatik sɛlɛkshɔn tɛmplat de jenarayz bay we dɛn de pik wit an bay we dɛn pik lɛk 250 piksɛl dɛn we gɛt 1000 patikyula dɛn insay 250 piksɛl freym ɛn nɔ rɛfrɛns tu-dimɛnshɔnal (2D) klasifikeshɔn, so dat dɛn de rijɛkt dɛn klasifikeshɔn dɛn we mit sampul kɔntaminɛshɔn ɔ we nɔ gɛt ɛni kwaliti we pɔsin kin no. Dɔn, dɛn bin du ɔtomɛtik sɛlɛkshɔn pan ɔl di maykrofoto dɛn, ɛn di RC-LH114-W na bin 849,359 patikyula dɛn, ɛn di RC-LH116 kɔmpleks na bin 476,547 patikyula dɛn. Ɔl di patikyula dɛn we dɛn dɔn pik dɔn pas tu rawnd dɛn we nɔ gɛt rɛfrɛns 2D klasifikeshɔn, ɛn afta ɛni rɔn, dɛn nɔ gri wit di patikyula dɛn we mit di kabɔn eria, sampul kɔntaminɛshɔn, nɔ klia ficha ɔ patikyula ɔvalap strɔng wan, we rili mek 772,033 (90.9%) ɛn 359,678 (75.5%) ) Dɛn yuz patikyula dɛn fɔ 3D klasifikeshɔn fɔ RC-LH114-W ɛn RC-LH116 rispɛktvɔli. Di initial 3D rεfrεns mכdel bin jεnarεt yuz di stochastik grεdiεnt dεsεnt mεtכd. Yuz di initial mכdel as rεfrεns, dεn kכl di patikyula dεm we dεn sεlekt insay fכ kεtכgrεf insay 3D. Yuz di mɔdel insay dis kategori as rɛfrɛns, du 3D rifinin pan di patikyula dɛn we de na di big kategori, dɔn yuz di initial 15Å lɔw-pas filta fɔ kɔba di sɔlvɛnt eria, ad 6 piksɛl dɛn fɔ sɔft edj dɛn, ɛn post-prɔsɛs di piksɛl dɛn fɔ kɔrɛkt di Gatan K2 pik Modulation transfa fɛnshɔn na di tɔp ditektɔ. Fכ di RC-LH114-W dataset, dis initial mכdel bin modify bay we dεn pul di strכng dεnsiti na di ed dεm fכ di mask (diskonekt frכm di kכr kכmpleks dεnsiti in UCSF Chimera). Di rizultin mכdel dεm (di rεsכlushכn dεm fכ RC-LH114-W εn RC-LH116 na 3.91 εn 4.16 Å, rispεktivli) dεn yuz as rεfrεns fכ di sεkכn raun כf 3D klasifikasiכn. Di patikyula dεm we dεn yuz dεn grup dεm insay di initial 3D klas εn dεn nכ gεt strכng kכrεleshכn wit di neba. Ɔvalap ɔ nɔ gɛt klia strɔkchɔral ficha dɛn. Afta di sɛkɔn raun fɔ 3D klasifikeshɔn, dɛn pik di kategori we gɛt di ays rizɔlt [Fɔ RC-LH114-W, wan kategori na 377,703 patikyula (44.5%), fɔ RC-LH116, tu kategori de, we totɛl 260,752 patikyula (54.7%) , Usay dɛn de di sem ɔl we dɛn alaynɛd ​​afta di fɔs rɔteshɔn wit smɔl difrɛns]. Dɛn kin pul di patikyula dɛn bak insay wan bɔks we gɛt 400 piksɛl ɛn dɛn kin rifin dɛn bay we dɛn de yuz 3D rifin. Di sכlvεnt mask de jεnarεt yuz di initial 15Å lכw-pas filta, 3 piksεl map εkspεnsכn εn 3 piksεl sכft mask. Yuz fɔ ɛni patikyula CTF rifinmɛnt, fɔ ɛni patikyula muvmɛnt kɔrɛkshɔn ɛn di sɛkɔn rawnd fɔ ɛni patikyula CTF rifinmɛnt, 3D rifinmɛnt, sɔlvɛnt mask ɛn post-prɔsɛsin dɛn de du afta ɛni stɛp fɔ rifin di tɛkstɔr we kɔmɔt mɔ. Yuz di FSC (Fourier Shell Correlation Coefficient) kot-ɔf valyu we na 0.143, di rizɔlt dɛn fɔ di fayn mɔdel dɛn fɔ RC-LH114-W ɛn RC-LH116 na 2.65 ɛn 2.80Å, rispɛktvɔli. Di FSC kɔv fɔ di fayn mɔdel de sho na Fig 2. S17.
כl di protin sikεns dεm dεn dכnכlod frכm UniProtKB: LH1-β (PufB; UniProt ID: Q6N9L5); LH1-α (PufA; YuniProtID: Q6N9L4); RC-L (PufL; YuniProt ID: O83005); RC-M (PufM; YuniProt ID: A0A4Z7); RC-H (PuhA; YuniProt ID: A0A4Z9); Protein-W (PufW; UniProt ID: Q6N1K3) we de mek yu gɛt di sik. SWISS-MODEL (45) wεn yuz fכ kכnstrכkt wan homכlכji mכdel fכ RC, we kכntεn di protin sikεns dεm fכ RC-L, RC-M εn RC-H εn di kristכl strכkchכ fכ Rba. sphaeroides RC bin yuz as tɛmplat (PDB ID: 5LSE) (46). Yuz di “fit map” tul insay UCSF Chimera fכ fit di mכdel we dεn jεnarεt to di map (47), impruv di protin strכkchכ, εn kכfaktכr [4×BChl a (monomer laybri rεsidyu nem = BCL), 2×BPh a (BPH), wan כ tu kayn UQ10 (U10), wan nכn-hεm ayכn (Fe) εn wan 3,4-dihaydrohexacarbonylcholine (QAK)] yuz Kut (48) fɔ ad. Sins QAK nɔ de na di monomer laybri, dɛn bin paramita am yuz di eLBOW tul na PHENIX ( 49 ).
Neks, di LH1 sabyunit bin kכnstrכkt. fכs, dεn yuz di כtomatכk kכnstrכkshכn tul na PHENIX (49) fכ כtomatכk kכnstrכkt pat pan di LH1 sikεns we yuz di map εn di LH1-α εn LH1-β protin sikεns dεm as input. sεlekt di mכst komplit LH1 sabyunit, εkstrakt am εn lod am insay Kut, ad di sikεns we nכ de insay wit yu an, εn manually rifin di כl strכkchכ bifo yu ad tu BCls a (BCL) εn wan spirilloxanthin (CRT) [akɔdin to di rilevεnt Rps Di dεnsiti fכ di LH1 kכmpleks εn di karotenoid kכntεnt we dεn no. Spisis (17)]. Kɔpi di kɔmplit LH1 sabyunit, ɛn yuz di UCSF Chimera “Docking Map Tool” fɔ dok na di adjasent nɔ-mɔdel eria fɔ LH1 density, ɛn afta dat rifin am na Coot; ripit di prכsεs te dεn mכdel כl di LH1 sεbyunit dεm. fכ di RC-LH114-W strכkchכ, bay we dεn de εkstrakt di unallocated density in di Coot, di protin de sεgmεnt frכm di rεmεnεnt nכn-protein kכmכpכnt dεm na di USCF Chimera map εn dεn yuz di Autobuild tul fכ establish di initial mכdel, εn di rεmεnεnt sכbyunit dεm (protein-W) Modeling. Insay FƐNIKS (49). Ad eni sikεns we mis to di rizultant mכdel in Coot (48), εn afta dat manually rifin di כl sabyunit. di rεmεnεnt unallocated dεnsiti fit di kכmbaynshכn כf lipid dεm (PDB mכnomεr laybri ID fכ CDL = CDL, POPC = 6PL εn POPG = PGT), β-DDM ditajεnt (LMT) εn UQ10 mכlikul dεm (U10). Yuz PHENIX optimization (49) ɛn manual optimization in Coot (48) fɔ pafɛkt di kɔmplit initial mɔdel te di mɔdel statystik ɛn di vijual kwaliti fɔ di fit nɔ go ebul fɔ impɔtant mɔ. Fɔ dɔn, yuz LocScale (50) fɔ shap di lokal map, ɛn afta dat, du sɔm ɔda saykl dɛn fɔ mɔdel di unallocated density ɛn ɔtomatik ɛn manual ɔptimayzeshɔn.
di rispεktiv pεptida dεm, kכfaktכ dεm εn כda lipid dεm εn kwinכn dεm we dכk insay dεn rispεktiv dεnsiti dεm dεn sho na fכg 1 εn 2. S18 to S23. Di statystik infɔmeshɔn fɔ di fayn mɔdel de sho na Tebul S1.
If dɛn nɔ tɔk ɔda we, dɛn bin gɛda di UV/Vis/NIR absɔpshɔn spɛktra pan Cary60 spɛktrofotomita (Agilent, USA) pan 1 nm intaval frɔm 250 nm to 1000 nm ɛn intagreshɔn tɛm we na 0.1s.
Dilute di sampul insay wan kwats kuvεt wit 2 mm pat to A880 כf 1, εn kכlekt di absכpshכn spεktrum bitwin 400 εn 1000 nm. Di sεkyulכr dichroik spεktra bin kכlekt pan wan Jasco 810 spεktropolarimeta (Jasco, Japan) pan 1 nm intaval bitwin 400 nm εn 950 nm pan wan skan rεt we na 20 nm min-1.
di molar extinction coefficient de ditarmin bay we yu dilute di kor komplex to wan A880 we de aprכksimatli 50. dilute di 10μl volyum insay 990μl binding buffer כ mεtanכl, εn kכlekt di absכpshכn spεktrum wantεm fכ minimiz BChl dεgradashכn. Di BChl kɔntinyu fɔ ɛni mɛtanɔl sampul bin kɔl bay di ɛkstinshɔn kɔfishin na 771 nm we na 54.8 mM-1 cm-1, ɛn dɛn bin disayd di ɛkstinshɔn kɔfishin ( 51 ). Divayd di BChl kɔnsɛntreshɔn we dɛn mɛzhɔ bay 32 (RC-LH114-W) ɔ 36 (RC-LH116) fɔ no di kɔr kɔmpleks kɔnsɛntreshɔn, we dɛn kin yuz afta dat fɔ no di absɔpshɔn spɛktrum fɔ di sem sampul we dɛn gɛda na di bafa Ɛkstinkshɔn kɔfishin. gri. Tri ripit mεjכmεnt bin tek fכ εvri sεmpl, εn di avεrej absכbכns כf di BChl Qy maksimכm bin yuz fכ kכlkyul. di εkstinkshכn kכfishεnt fכ RC-LH114-W we dεn mכsu na 878 nm na 3280±140 mM-1 cm-1, we di εkstinkshכn kכfishεnt fכ RC-LH116 we dεn mεzj na 880 nm na 3800±30 mM-1 cm-1.
UQ10 bin kwantifay akɔdin to di we aw dɛn de du am na (52). In shot, rivεs fεz HPLC (RP-HPLC) bin pεrform yuz di Agilent 1200 HPLC sistεm. Sɔlv lɛk 0.02 nmol RC-LH116 ɔ RC-LH114-W insay 50μl 50:50 mɛtanɔl:klorofɔm we gɛt 0.02% (w/v) fɛrik klorayd, ɛn injɛkt di prɛ-ɛkwilibret Beckman Coulter Ultrasphere ODS 4.6 mm Sɔlv insay 1 ml-1 min-1 na 40°C insay HPLC sכlvεnt (80:20 mεtanכl: 2-prכpanכl) pan wan ×25 cm kכlכm. Pεrform isocratic elution in wan HPLC sכlvεnt fכ mכnitor absכbכns na 275 nm (UQ10), 450 nm (karotenoyd) εn 780 nm (BChl) fכ 1 awa. di pik in di 275 nm kromatogram na 25.5 minit bin intagret, we nכ bin kכntεn nכ כda dεtektabl kכmpawnd. di intagreted eria de yuz fכ kכl di molar amoun fכ UQ10 we dεn εkstrakt wit rεfrεns to di kalibrayshכn kεrv we dεn kכl frכm di injεkshכn fכ pure standad frכm 0 to 5.8 nmol (Figure S14). Dɛn bin analayz ɛni sampul insay tri riplikɛt dɛn, ɛn di mistek we dɛn ripɔt kɔrɛkt to di SD fɔ di avɛrej.
A sכlushכn we kכntεn di RC-LH1 kכmpleks wit maksimal Qy absכpshכn we na 0.1 bin pripia wit 30 μM ridyus כs hat saytokrכm c2 (Merck, UK) εn 0 to 50 μMUQ2 (Merck, UK). Tri 1-ml samplε dεm bin pripia pan εvri UQ2 kכnsantreshכn εn inkכba כvnayt insay dak na 4°C fכ mek sכh se komplit adaptεshכn to di dak bifo mεzjכ. Di solushɔn bin lod insay wan OLIS RSM1000 modular spɛktrofotomita we gɛt 300 nm flaym/500 layn gretin, 1.24 mm inlɛt, 0.12 mm midul ɛn 0.6 mm ɔlt slit. Dɛn put wan 600 nm lɔng pas filta na di ɛntrɛ na di sampul fototyub ɛn di rɛfrɛns fotomultiplaya tyub fɔ mek dɛn nɔ gɛt ɛksayteshɔn layt. Di absכrbans bin mכnitor na 550 nm wit intagrεshכn tεm we na 0.15 s. Di ɛksayteshɔn layt de kɔmɔt frɔm di 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) tru wan fayba ɔptik kebul we gɛt 90% intensiti tru wan DC2200 kɔntrol (Thorlabs Ltd., UK) ɛn i de kɔmɔt to di layt sɔs pan wan angul we na 90° ɔf. Di mɛzhɔmɛnt bim de agens di miro fɔ mek i kam bak wit ɛni layt we di sampul nɔ bin absɔb fɔs. Monitor di absorbans 10 s bifo di iluminans 50 s. afta dat, dεn bin mכnitor di absכbεns fכ 60 s insay di dak fכ ases di εkstenshכn we kwinolכl spontan rεdכks saytokrכm c23 + (luk fכg S8 fכ raw data).
Di data bin prosɛs bay we dɛn fit wan linya initial rit insay 0.5 to 10 s (dipen pan di UQ2 kɔnsɛntreshɔn) ɛn avɛrej di rit dɛn fɔ ɔl di tri sampul dɛn na ɛni UQ2 kɔnsɛntreshɔn. Di RC-LH1 kɔnsɛntreshɔn we dɛn kɔl bay di rispɛktiv ɛkstinshɔn kɔfishin bin yuz fɔ kɔnvɔyt di rit insay di katalaytik efyushɔn, we dɛn plot insay Origin Pro 2019 (OriginLab, USA), ɛn fit am to di Michaelis-Menten mɔdel fɔ no di apɔrint Km ɛn Kcat valyu dɛn.
Fכ transiεnt absכpshכn mεjכmεnt, di RC-LH1 sεmpl bin diluted to ~2μM in IMAC bכfa we kכntεn 50 mM sכdyכm askorbεt (Merck, USA) εn 0.4 mM Terbutin (Merck, USA). askorbik asid dεn de yuz am as sakrifays εlektrכn dכna, εn dεn de yuz tεrt-butaklofen as QB inhibito fכ mek sכh se di men RC dכna de rεdכks (dat na, nכ fכto כksidayz) tru di mεzhɔmεnt prכsεs. aprכksimatli 3 ml sεmpl dεn ad to wan kכstכm rotatin sεl (bכt 0.1 m in dayamita, 350 RPM) wit 2 mm optik pat lεngth fכ mek sכh se di sεmpl na di lεzεr pat gεt inof tεm fכ dak adapteshכn bitwin εksayz puls dεm. Yuz ~100-fs laser puls fɔ amplify di Ti: Sapphire laser sistem (Spectra Physics, USA) fɔ ɛksayt di sampul na 880 nm pan ripitishɔn rit we na 1 kHz (20 nJ fɔ NIR ɔ 100 nJ fɔ Vis). Bifo yu gɛda di data, put di sampul pan ɛksayteshɔn layt fɔ lɛk 30 minit. εkspכzכn go mek QA inaktivεshכn (possibly ridyus QA wan כ tu tεm). Bɔt duya notis se dis prɔses kin chenj bikɔs afta lɔng tɛm we dɛn dɔn adap to dak, RC go go bak smɔl smɔl to QA aktiviti. A Helios spɛktromita (Ultrafast Systems, USA) bin yuz fɔ mɛzhɔ transiɛnt spɛktra wit dilɛy tɛm we na -10 to 7000 ps . Yuz Surface Xplorer softwe (Ultrafast Systems, USA) fɔ ungroup di data sɛt dɛn, dɔn dɛn jɔyn ɛn standad. Yuz di CarpetView softwea pakej (Light Conversion Ltd., Lithuania) fɔ yuz di kɔmbayn data sɛt fɔ gɛt difrɛnshal spɛktra dɛn we gɛt fɔ du wit dɛkayshɔn, ɔ yuz wan fɛnshɔn we de kɔnvɔl bɔku ɛkspɔnɛnt dɛn wit di inschrumɛnt rispɔns fɔ fit di singl-wevlɛnj spɛktral ɛvolushɔn insay Ɔrijin (OriginLab, USA).
as wi bin mεnshכn oba (53), dεn bin pripia wan fכtosεntetik film we kכntεn LH1 kכmpleks we nכ gεt כl tu di RC εn pεrifεral LH2 εntena. Di membran bin diluted in 20 mM tris (pH 8.0) ɛn afta dat dɛn lod am insay wan kwats kuvɛt wit 2 mm ɔptik pat. A 30nJ laser puls wεn yuz fכ εksayz di sεmpl na 540 nm wit dilay tεm we -10 to 7000 ps. Proses di data set as dɛn diskrayb am fɔ Rps. pal sampul.
Di membran bin pεlεt bay sεntrifugayshכn na 150,000 RCF fכ 2 awa na 4°C, εn afta dat in absכbεns na 880 nm bin rεsuspεnd insay 20 mM tris-HCl (pH 8.0) εn 200 mM NaCl. Sɔlv di mɛmbran bay we yu de stɔp smɔl smɔl insay 2% (w/v) β-DDM fɔ 1 awa na dak na 4°C. Di sampul bin dil insay 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) to wan protin kɔnsɛntreshɔn we na 2.5 mg ml-1 (Bio-Rad analisis). fכda prכsεsin bin kכriכnt frכm di mεtכd we dεn bin pכblish bifo ( 54 ), stat wit di dilushכn כf 50 μg protin insay wan tכtal 50 μl TEAB we kכntεn 1% (w/v) sכdyכm laurate (Merck, UK). Afta sonication fɔ 60 s, i bin ridyus wit 5 mM tris (2-carboxyethyl) phosphine (Merck, UK) na 37 ° C fɔ 30 minit. Fכ S-alkylation, inkubayt di sεmpl wit 10 mM mεtil S-methylthiomethanesulfonate (Merck, UK) εn ad am frכm 200 mM isopropanol stok sכlushכn fכ 10 minit na rum tεmprachכ. Proteolytic digestion bin kכriכnt bay we dεn ad 2 μg trypsin/endoproteinase Lys-C miksכp (Promega UK) εn inkכba am na 37°C fכ 3 awa. Di laurate surfactant bin ekstrakt bay we dɛn ad 50 μl ethyl acetate ɛn 10 μl 10% (v/v) LC gred trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) ɛn vortex fɔ 60 s. Di fεz separeshכn bin prכmot bay sεntrifugayshכn na 15,700 RCF fכ 5 minit. akɔdin to di manifakta in protɔkɔl, dɛn yuz wan C18 spin kɔlɔm (Thermo Fisher Scientific, UK) fɔ tek tɛm aspirayt ɛn disɔlt di lɔwa faz we gɛt di peptide. Afta dray bay vaykum sɛntrifugayshɔn, di sampul bin sɔlv insay 0.5% TFA ɛn 3% asɛtonitril, ɛn 500 ng bin analayz bay nanoflɔ RP kromatografi kɔpl wit mas spɛktrometri yuz di sistɛm paramita dɛn we dɛn bin dɔn tɔk bɔt bifo tɛm.
Yuz MaxQuant v.1.5.3.30 (56) fכ protin idεntifikεshכn εn kwantifayshכn fכ sכch fכ Rps. palustris proteom dεtabεys (www.uniprot.org/proteomes/UP000001426). Di mas spɛktromɛtri proteɔmiks data dɔn de na di ProteomeXchange Alliance tru di PRIDE patna ripɔsitɔri (http://proteomecentral.proteomexchange.org) ɔnda di dataset aydentifaya PXD020402.
Fɔ analisis bay RPLC kɔpl wit ilɛktrɔsprɛy ayɔnizashɔn mas spɛktrometri, di RC-LH1 kɔmpleks bin pripia frɔm wayl-tayp Rps. Yuz di mεtכd we dεn bin pכblish bifo (16), di protin kכnsantreshכn we dεn prodyuz insay palustris sεl dεm na bin 2 mg ml-1 insay 20 mM Hepes (pH 7.8), 100 mM NaCl εn 0.03% (w/v) β- (Bio-Rad analisis) ) DDM. Akɔdin to di manifakta in protɔkɔl, yuz 2D purifyeshɔn kit (GE Healthcare, USA) fɔ pul 10 μg protin bay prɛsipitashɔn we, ɛn sɔlv di prɛsipitayt insay 20 μl 60% (v / v) fɔmik asid (FA), 20% (v / v) Asɛtonitril ɛn 20% (v/v) wata. Fayv maykrolit dɛn bin analayz bay RPLC (Dionex RSLC) we dɛn kɔpl wit mas spɛktrometri (Maxis UHR-TOF, Bruker). Yuz MabPac 1.2×100 mm kɔlɔm (Thermo Fisher Scientific, UK) fɔ separeshɔn na 60°C ɛn 100μlmin -1, wit wan gradient we na 85% (v / v) sɔlvɛnt A [0.1% (v / v) FA ɛn 0.02% (V/v) TFA akyuɔs sɔlvushɔn] to 85%(v/v) sɔlvɛnt B [0.1%(v/v) FA ɛn 0.02%(v/v) insay 90%(v/v) asɛtonitril TFA] Yuz standad ilɛktrɔsprɛy ayɔnizashɔn sɔs ɛn difɔlt paramita fɔ mɔ pas 60 minit, di mas spɛktromita de gɛt 100 to 2750 m/z (mas-to-chaj rεshɔn). Wit di ɛp fɔ di ExPASy bioinformatics risɔs pɔtal FindPept tul (https://web.expasy.org/findpept/), map di mas spɛktrum to di sabyunit dɛm na di kɔmpleks.
Di sel dεm bin gro fכ 72 awa כnda 100 ml NF-lכw (10μMm-2 s-1), mεdiכm (30μMm-2 s-1) כ hεy (300μMm-2 s-1) layt. M22 mεdiכm (M22 mεdiכm we dεn kכl amoniכm sכlfεt εn sכdiכm sכksinayt de riples wit sכdiכm asetεt) insay 100 ml skru-tכp bכtul (23). In fayv 30-s saykl, 0.1 maykron glas bidz bin biad pan volyum rεshכn 1: 1 fכ layz di sεl dεm εn kol pan ays fכ 5 minit. Di tin we nɔ sɔlv, di sɛl dɛn we nɔ brok ɛn di glas bidz dɛn bin pul am bay we dɛn yuz sɛntrifugayshɔn na 16,000 RCF fɔ 10 minit insay wan bɛnchtɔp maykrosɛntrifuj. Di membran bin separet insay Ti 70.1 rɔta wit 100,000 RCF insay 20 mM tris-HCl (pH 8.0) wit 40/15% (w/w) sukrɔs gradient fɔ 10 awa.
as dεn dεskrεb insay wi fכs wok, imyunodεkshכn fכ di His tεg pan PufW ( 16 ). in shot, di purified kor komplex (11.8 nM) כ di mεmbran we kכntεn di sem kכnsantreshכn fכ RC (dεn dεtermin bay כksidεshכn we de sכbtrakt di ridyus difrεns spεktrum εn mach di lod pan di stεyn jel) insay 2x SDS lodεn bכfa (Merck, UK) we dεn dil tu tεm. Di protin dεm bin separet pan wan rεplik 12% bis-tris NuPage jel (Thermo Fisher Scientific, UK). A jel bin stɛyn wit Coomassie Brilliant Blue (Bio-Rad, UK) fɔ lod ɛn visualize di RC-L sabyunit. di protin we de pan di sεkכn jel bin transfכm to mεtanכl-aktivεt polyvinylidene fluoride (PVDF) mεmbran (Thermo Fisher Scientific, UK) fכ immunoassay. Di PVDF membran bin blok insay 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 ɛn 5% (w / v) skim milk pauda, ​​ɛn afta dat dɛn inkubayt am wit di anti-His praymari antibodi (in Dilute di antibodi bafa [50 mM tris-HCl (pH 7.6), 150 mM NaCl ɛn 0.05% (v/v) Tween-20] insay 1:1000 A190-114A, Bethyl Laboratories, USA) fɔ 4 awa. Afta yu was 3 tεm fכ 5 minit insay antibodi bafa, dεn kכmbayn di mεmbran wit hכsεradish pεrכksidεz (Sigma-Aldrich, UK) anti-maus sεkכnd antibodi (diluted 1:10,000 in antibodi bafa) Inkubayt fכ alaw dεtekshכn (5 minit afta 3 was insay antibodi bafa) yuz WESTAR ETA C 2.0 kεmiluminescence sabstεrayt (Cyanagen, Itali) ɛn Amersham Imej 600 (GE Ɛlthkɛr, UK).
bay we yu drכ di intensiti distribushכn fכ εvri steyn jel כ immunoassay len, intagret di εria כnda di pik εn kכl di intensiti rεshכn fכ RC-L (stained jel) εn Protein-W (immunoassay), insay ImageJ (57) Proses di imej. Dis rεshכn dεm bin kכnvכlt to molar rεshכn bay we dεn asum se di rεshכn fכ RC-L to protin-W insay di pure RC-LH114-W sεmpl na 1:1 εn nכmal di כl data sεt akɔdin to dat.
Fɔ sɔpɔt ɔda tin dɛn fɔ dis atikul, duya luk http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
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di ay-rεsכlushכn strכkchכ fכ di layt trap 1 kכmpleks na di riakshכn sεntr de gi nyu insayt dεm fכ di kwinכn dinamiks.
Devid J. K. Swɛnsbɔri, Pak Qian, Filip J. Jaksin, Keytlin M. Faries, Dariusz M. Niedzwiedzki, Ɛlizabɛt C. Matin, Devid A. Fama, Lɔrna A. Malɔn, Ribɛka F. Tɔmsin, Nil A. Ransin, Daniɛl P Kanif , Mak J. Dikman, Duy Ɔltɛn, Kristin Kirmaier, Andru Hitchkɔk, C. Nil Hɔnta
di ay-rεsכlushכn strכkchכ fכ di layt trap 1 kכmpleks na di riakshכn sεntr de gi nyu insayt dεm fכ di kwinכn dinamiks.
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