Natan†Kaplesan ya address: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Say reaction center light-harvesting complex 1 (RC-LH1) et manunan kabiangan na photosynthesis na purple phototrophic bacteria. Inloob mi so duaran istruktura na cryo-electron microscopy na RC-LH1 complex manlapud Rhodopseudomonas palustris. Say 2.65-Å ya resolusyon ya istruktura na RC-LH114-W complex et tugyopen na 14 subunit LH1 loops ya akapaliber ed RC, ya nababalang na protina W, bangta say complex ya anggapoy protina-W et sigpot ya RC komposisyon ya akapaliber ed RC. Insara so 16 subunit LH1 loop. Say pangikompara ed sarayan istruktura et mangiter na pakatalos ed dinamika na quinone ed RC-LH1 complex, kaiba lay ag ni nadetermina iran pananguman ed konpormasyon no manbibiig na quinone ed RC QB site, ontan met ed kawalaan na saray auxiliary quinone binding sites, ya ontutulong pian nipasa iratan ed RC. Say nikadkaduman istruktura na W protein et mangaamper ed pansara na LH1 loop, kanian makagawa na channel parad pamapeles na quinone/quinolone exchange.
Say enerhiyan itatarya na photosynthesis et makapansiansia ed ngalngalin amin a bilay ed dalin, tan baleg so potensyal to parad solar biotechnology. Legan ya ipapaseseg so sankamundoan ya photosynthesis, say purple phototrophic bacteria et mangipapatnag met na nanduruman paraan na enerhiya tan abilidad ed metabolismo. Sarag dan paliisan so photosynthesis tan ombaleg ira bilang heterotrophic bacteria ed ambilunget, sarag dan i-fix so nitrogen tan carbon dioxide, makapawala na hydrogen, tan makaderal na aromatic compounds (1-3). Pian nawalaan na enerhiya irayan proseso, say liwawa et kaukolan ya maples tan epektibon magmaliw ya kemikal ya enerhiya. Sayan proseso et onggapo no say light-trapping antenna complex et mansepsep na liwawa tan iyalis to so naala ya enerhiya ed reaction center (RC), diad ontan et onggapo so pansian na karga (4 – 7). Say manunan yunit na photosynthesis ed purple phototrophic bacteria et tugyopen na type 2 RC, ya apaliberan na light-harvesting complex 1 (LH1), ya manporma na RC-LH1 core complex. Say LH1 et naporma na sakey a array na kurbadon αβ heterodimers, ya balang sakey ed saratan et mangibibiig ed duaran bakterial chlorophyll (BChl) a molecules tan sakey odino duaran carotenoids (8-12). Say sankasimplian ya LH1 antenna et tugyopen na 16 o 17 αβ heterodimers ya akapaliber ed RC (9-13) diad sakey a saradon loop, balet diad arum ya core complexes, saray transmembrane peptides so mangaamper ed pantultuloy na LH1 ya akapaliber, Diad ontan et napaaligwas so quinol/quinone diffusion ed baetan na RC tan cytochrome bc complex (1111, 13). Say purple phototrophic ya tanaman ya Rhodopseudomonas (Rps.) et sakey a modelon organismo ya makatalos ed enerhiya tan electron transfer ya onsusuporta ed photosynthesis. Say inmunan kristal ya istruktura na Rps. Say modelo na palustris RC-LH1 complex et RC, ya apaliberan na 15 ya heterodimeric LH1 loops, ya nababalang na ag kabat ya protina ya tatawagen ya “Protein W” (14). Say Protein-W et abidbir bilang RPA4402, ya sakey ya ag-amta ya 10.5kDa ya protina ya walaan na taloran naisip ya transmembrane helices (TMH) (16). Iproproponin umanen so ngaran na rpa4402 gene ya man-encode na protina W ed pufW pian mipara ed nomenklatura ya inusar parad saray genes ya man-encode na RC-L, M (pufL, pufM) tan LH1α, β (pufA, pufB) subunits. Makapainteres, say protein-W et wala labat ed manga 10% na RC-LH1, ya mangipapatnag na Rps. say palustris et manggagawa na duaran nanduruman RC-LH1 complex. Dia, irereport mi so high-resolution ya cryo-EM (cryo-EM) ya istruktura na duaran manunan komplikado, sakey ya walay protina W tan 14 αβ heterodimers, say sananey et anggapoy protina W tan sakey ya sarado ya 16 Heterodimer LH1 loop. Say istruktura tayo et mangirerepresenta na pananguman ed pakatalos ed RC-LH1 complex na Rps. palustris, lapud inanalisa mi so pareparehon populasyon na kada variant tan walaan kami na magenap a resolusyon pian malinew ya nibiig so kada peptide tan saray akapeket a pigment tan saray misiglaotan a lipid tan quinones. Say pangikompara ed sarayan istruktura et mangipapanengneng ya say taloran TMH proteins-W ya ag ni aromog ed arum ya RC-LH1 complex anggad natan et manggagawa na quinone channel pian napapeles so quinone/quinolone exchange. Walay pigaran napreserba ya lipid tan quinone binding sites ya abitla, tan walay balon pananguman ed konpormasyon kayari na kombinasyon na quinone tan RC, ya nayarin matukoy parad photosystem II (PSII) RC na saray oxygenated phototrophic organisms. Say adiskobre mi et mangiiter na balon pakatalos ed kinetics na quinone/quinolone binding tan exchange ed RC-LH1 core complex na purple phototrophic bacteria.
Pian napagaan so detalyadon panaral ed duaran komplikadon naromog ed Rps. palustris, ibibiig mi so kada RC-LH1 panamegley na biochemical iran paraan. Say protein W-deficient complex (ya tatawagen ya ΔpufW) et pinuran manlapud strain ya anggapoy pufW gene (16), tan sakey labat ya RC-LH1 complex so nayarin nagawa. Say protein W-containing complex et napapawala na sakey a strain. Say protina W na sayan strain et inuman na 10x His tag ed C-terminus to, pian say protina W-containing complex et epektibon natipon ed karaklan ya kulang na protina W diad pangi-immobilize ed metal. Say komplikado et epektibon naapag (16) Affinity Chromatography (IMAC).
Unong ed nanengneng ed Figure 1, parehon walaan na taloran sub-unit RC (RC-L, RC-M tan RC-H) ya apaliberan na LH1 antenna. Say 2.80-A ya istruktura na complex ya anggapoy protein-W et mangipapanengneng na 16 αβ heterodimers, ya manporma na saradon LH1 loop ya sigpot ya akapaliber ed RC, ya tatawagen ya RC-LH116 complex. Say 2.65Å ya istruktura na protein-W-containing complex et walaan na 14-heterodimer LH1 ya intunda na protein-W, ya tatawagen ya RC-LH114-W.
(A tan B) Representasyon na tapew na compound. (C tan D) Saray nankakasakey a kolor ya nipatnag ed saray baras. (E tan F) Saray komplikado ya naobserbaan manlapud tapew na cytoplasm et walaan na saray peptide tan LH1 subunits ya nirepresenta ed saray cartoon, tan nibilangan ira ya ontutumbok ed relo manlapud protein-W gap [ya mipara ed Rba numbering. sphaeroides complex (13)]. Parad LH1-α, say kolor na subunit na protina et duyaw; parad LH1-β, say kolor na subunit na protina et asul; parad protina-W, say protina et ambalanga; parad RC-H, cyan itan; parad RC-L, Orange itan; parad RC-M, magenta. Saray cofactors et irerepresenta na saray baras, say berde et irerepresenta na BChl tan BPh a molecules, say purpura et irerepresenta na carotenoids, tan say duyaw et irerepresenta na UQ10 molecules. (G tan H) Pinabaleg a nengnengen so protein-W gap ed mipara a rehyon na RC-LH114-W complex (G) tan RC-LH116 complex (H). Saray cofactors et nipanengneng ed nengneng na space filling, say chelated quinone et nipanengneng ed asul. Say protein-W gap et nipabitar na asul ya putol-putol a linya ed (G), tan saray melag ya abut ya ditan so panlalapuan na quinone/quinolol ed LH116 ring et nipabitar na andeket ya putol-putol a linya ed (H).
Ipapanengneng na Figure 1 (A tan B) so RC ya apaliberan na lukas odino sarado iran array na LH1αβ heterodimers, ya balang sakey et manbibiig na duaran BChl tan sakey a carotenoid (Figure 1, C tan D). Impanengneng na saray akaunan impanaral ya say Rps et say LH1 complex. Diad biosynthetic pathway na spirulina xanthin, sarayan klase na tanaman et walaan na nanduruman populasyon na carotenoids (17). Balet, say spiropyrroxanthin so manunan carotenoid tan say karakel to et makapenek. Kanian, pinili min imodelo so spiroxanthin ed amin ya LH1 binding sites. Say alpha tan beta polypeptides et saksakey ya TMH ya walay antikey ya membrane ed paway na rehyon (Figure 1, A, B, E, tan F). Anggaman ag-anengneng so karakel na 17 ya residue ed C-terminus, say alpha polypeptide et apaldua manlapud Met1 ya anggad Ala46 ed parehon complex. Say β polypeptide et nabawasan manlapud Gly4 ya anggad Tyr52 ed RC-LH116, tan manlapud Ser5 ya anggad Tyr52 ed RC-LH114-W. Anggapoy naobserbaan ya densidad na 3 o 4 N-terminal o 13 C-terminal residues (Figure S1). Say mass spectrometry analysis na mixed RC-LH1 complex ya imparaan manlapud wild-type strain et angipatnag ya say kulang ya rehyon et resulta na heterologous cleavage na sarayan peptides (Figure S1 tan S2). Naobserbaan met so N-terminal formylation na α-Met1 (f). Impanengneng na analysis ya say α-peptide et tugyopen na residues fMet1 anggad Asp42/Ala46/Ala47/Ala50, tan say β-peptide et tugyopen na residues Ser2 anggad Ala53, ya mipara ed low-temperature EM density map.
Say koordinasyon na α-His29 tan β-His36 so manggawa ed saray BChls ya arap-arapan; kada αβ heterodimer et mantitipon ed saray kakaabay to pian manporma na lukas a loop (RC-LH114-W) odino sakey a sarado a loop (RC-LH116) ed kaliberliber na RC Say exciton coupled pigment array (Figure 1, C tan D). No ikompara ed 877 nm ya banda na RC-LH114-W, say 880 nm ya absorption red shift na RC-LH116 et 3 nm (Figure 2A). Balet, say circular dichroism spectrum et ngalngalin pareho (Figure 2B), ya mangipapanengneng ya anggaman walay malinew ya pandumaan na open tan closed loops, say lokal ya kaliberliber na BChls et mipara. Say absorption redshift et nayarin resulta na nabawasan ya thermal motion tan indakel na stability ed closed loop (18, 19), say panguman na pigment coupling ya sengegan na closed loop (20, 21), odino say kombinasyon na sarayan duaran epekto (11).
(A) Ultraviolet/visible/near-infrared absorption spectrum, ya saray peak to et akamarkaan na saray mipetekan ya pigment da tan in-normalize ed BPh peak ed 775 nm. (B) Circular dichroism spectrum ya innormalize ed BChl absorbance ed 805 nm. (C tan D) Napili so ΔA spectra manlapud time-resolved absorption spectra na RC-LH114-W complex (C) tan RC-LH116 complex (D). Parad mas maabig ya panangikompara, amin ya spectra et na-normalize ed ∆A na −A ed 0.2 ps. (E) Say peles na cytochrome c2 oxidation kayari na irradiation ed kiwawala na nanduruman konsentrasyon na UQ2 (nengnengen so Figure S8 parad raw data). (F) Diad saray selula ya binmaleg ed silong na abeban, kapegleyay odino atagey ya intensity na liwawa (10, 30 o 300μMm-2 s-1, unong ed inkauksoy da), say protina W tan RC-L subunits ed purified complex tan say separated membrane ratio. Amtaen so lebel na protina panamegley na SDS-polyacrylamide gel electrophoresis tan immunoassay (nengnengen so Figure S9 parad raw data). Amtaen so ratio ya mipara ed puron RC-LH114-W complex. Say stoichiometric ratio na RC-L ed protein-W na complex et 1:1.
Saray BChls ed posisyon 1 ed deformed αβ14 loop na RC-LH114-W (Figure 1, A, C, tan E) et mas asingger ed RC primary donor (P) na 6.8Å nen saray mipara ya BChls ed RC-LH116 (Figure 1, B, D, tan F, tan S3); balet, say transient absorption kinetics na duaran complex so mangipapanengneng ya parad RC-LH114-W tan RC-LH116, say excitation energy transfer time constants manlapud LH1 ya anggad RC et 40 ±4 tan 44±3 ps (Figure 2). , C tan D, Figure S4 tan Table S2). Anggapo met so signipikantin pandumaan na electronic transfer ed loob na RC (Figure S5 tan saray mikonektaan ya supplementary text). Iisipen mi ya say maapit a pantutunosan na panaon na pangiyalis na enerhiya ed baetan na LH1 tan RC-P et lapud parehon distansya, anggulo tan potensyal ya enerhiya na karaklan ya BChl ed duaran LH1 loop. Ompatnag ya say pansukimat ed LH1 energy pattern pian naabot so sankaabebaan a distansya et aliwan mas maples nen say direktan pangiyalis na enerhiya manlapud saray suboptimal a pasen ya anggad RC. Say open-loop LH1 loop ed RC-LH114-W et nayarin ondalan met na ag tanton thermal motion ed silong na abeban temperatura parad structural analysis, tan walay mas andukey ya αβ14 ring conformation ed temperatura na kuarto manlapud pigmentation distance na βBChls diad posisyon na RC 1.
Say RC-LH116 complex et walaan na 32 BChls tan 16 carotenoids, tan say inkalapagan ya inkauksoy to et pareho ed samay naala ed Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) DB strain (PDB) ID 5Y5S) (97) tan green algae (Blc.viridis) (PDB ID 6ET5) (10). Kayari na impan-align, melag labat so anengneng ya iyaamot ed posisyon na αβ heterodimers, lalo la ed 1-5, 15, tan 16 (Figure S6). Say kiwawala na protina-W et walay baleg ya epekto to ed istruktura na LH1. Say taloran TMH to et mankokonektaan panamegley na antikey iran loop, ya say N-terminal et walad gilig na lumen na complex tan say C-terminal et walad gilig na cytoplasmic (Figures 1A tan 3, A anggad D). Say Protein-W et maslak ya hydrophobic (Figure 3B), tan say TMH2 tan TMH3 et mibabali ed LH1αβ-14 pian manporma na transmembrane surface (Figure 3, B tan E anggad G). Say interface et manunan tugyopen na Phe, Leu tan Val residues ed transmembrane region. Sarayan residue et aka-stack na hydrophobic amino acids tan αβ-14 pigments. Wala ray polar residues ya ontutulong met ed pidedeneng, kaiba lay hydrogen bond ed baetan na W-Thr68 tan β-Trp42 diad tapew na komplikadon lubok (Figure 3, F tan G). Diad tapew na cytoplasm, say Gln34 et asingger ed keto group na αβ-14 carotenoids. Niarum ni, say n-dodecyl β-d-maltoside (β-DDM) ya molekula et naresolbe, tan say hydrophobic tail to et inmabot ed interface na protein-W tan αβ-14, tan say lipid tail et nayarin walad laman. Naimano mi met ya saray C-terminal resolution regions na protein W tan RCH et asingger a tuloy, balet aliwan diad loob na sakop na panporma na espisipikon interaksion (Figure 1, A tan E). Balet, nayarin wala ray pidedeneng ed saray agni naresolbe ya C-terminal amino acid na sarayan duaran protina, ya nayarin mangiter na mekanismo parad panag-recruit na protina-W legan na panagtipon na RC-LH114-W complex.
(A) Say protein-W, ya akaarap ed interface na LH1αβ14 ya singa cartoon, et walay rod-shaped side chain (ambalanga), ya nanengneng ed sakey a parte na electrostatic potential diagram (transparent gray surface ya walay contour level ya 0.13). (B) Protein-W ya irerepresenta na sakey a kolor ya tapew ya ag-onkukurang ed danum. Saray polar tan charged ya lugar et nipanengneng ed cyan, saray hydrophobic ya lugar et nipanengneng ed amputi, tan saray mabiskeg ya hydrophobic ya lugar et nipanengneng ed orange. (C tan D) Say protina-W ya irerepresenta ed cartoon, say orientation to et pareho ed (A) (C), tan inpaliber na 180° (D). Unong ed posisyon ed nantutumbokan, saray residue ya nabidbir et walaan na kolor ya rainbow, ya say N-terminal et asul tan say C-terminal et ambalanga. (E) Protein-W ed parehon nengnengen a singa ed (A), tan saray residue ed interface na protein-W:LH1 et nirepresenta na saray baras ya walay akapeket iran marka. (F) Say Protein-W et in-rotate na 90° no ikompara ed (E) tan LH1αβ14 diad representasyon na cartoon, tan no ikompara ed saray interface residues ed representasyon na bar. Saray akasabit ya residue manlapud beta polypeptide et walay label to. Say cofactor et nipanengneng bilang bar ya mipara ed kolor na Figure 1, say abulok ya β-DDM et nipanengneng ed abuek, tan say oxygen et nipanengneng ed ambalanga. (G) Say nengnengen ed (F) et in-rotate na 180°, a walaan na saray prominentin residue na aka-label ya alpha polypeptide.
Say Protein-W et onsalat ed αβ heterodimer (say koma-15 ed Figure 1F), kanian ag-onsara so loop tan onkiling so unonan taloran αβ heterodimer. Naobserbaan ya say sankatageyan ya anggulo na inklinasyon na unonan αβ-1 heterodimer no ikompara ed normal na pelikula et 25° anggad 29° (Figure 1, A tan E), ya aporma na 2° anggad 8° ya inklinasyon na αβ-1 ed RC A sharp contrast-LH116 (Figure 1, B tan F). Say komadua tan komatlon heterodimers et aka-inclin ed 12° anggad 22° tan 5° anggad 10°, unong ed inkauksoy da. Lapud steric hindrance na RC, say tilt na αβ-1 et ag to kaiba so komaduan paris na αβ (ya mipara ed koma-16 ya αβ ed Figure 1F), kanian walay malinew ya gap ed LH1 ring (Figure 1, A tan E). Lapud anggapoy duaran αβ heterodimers, ya kaiba so inkaandi na apatiran BChl tan duaran carotenoids, anggapo ed saray carotenoids so onsisiglaot ed twisted αβ-1 subunit, kanian walay LH114-W ring ya walaan na 13 carotenoids Vegetarian tan 28 BChls. Say lokal ya kalkulasyon na resolusyon na duaran komplikado ed αβ1 anggad 7 a rehyon et abeba nen saray arum nin kabiangan na LH1 loop, ya nayarin mangipapatnag na inherent plasticity na LH1 subunit ya asingger ed RC QB site (Figure 4) .
Saray litrato na RC-LH114-W (A tan B) tan RC-LH116 (C tan D) et nipanengneng manlapud parehon top view/side view (A tan B) (A tan C) tan cavity surface na Fig. 1. (B tan D). Saray kolor ya tombok et nanengneng ed nikawanan.
Say sananey nin karakter ya core complex ya walay stoichiometric ratio ya 1:14 et say Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Balet, say protina W tan PufX et anggapoy malinew ya pankakasakey da, tan walay baleg ya epekto ra ed saray istruktura na LH1 da. Say PufX et sakey a TMH ya walay N-terminal cytoplasmic domain ya mibabali ed cytoplasmic side na RC-H subunit (13) diad posisyon ya mipara ed Rps. palustris LH116αβ-16. Say PufX et manggagawa na channel parad quinone/quinolone exchange ed baetan na RC-LH1 tan say cytochrome bcl complex tan walad amin ya Rba. sphaeroides core complex (13). Anggaman say monomer-monomer interface et walad Rba. Say sphaeroides RC-LH1-PufX dimer et walad kawalaan na protina W ed RC-LH114-W, tan say agwat ya sengegan na PufX tan protina-W et walad parehon posisyon (Figure S7A). Say gap ed RC-LH114-W et mipara met ed hypothetical quinone channel (8) na Pseudomonas rosea LH1, ya naporma na saray peptide ya ag mikonektaan ed protina W o PufX (Figure S7B). Niarum ni, say quinone channel ed Blc. Say emerald green LH1 ya aporma lapud ag-inlaktip so sakey ya γ subunit (7) et walad mipadparan posisyon (Figure S7C). Anggaman nandurumay protina ya ipapaarap, say itsura na sarayan quinone/quinolol channels ed parehon posisyon ed RC-LH1 complex et singa sakey ya alimbawa na convergent evolution, ya mangipapanengneng ya say gap ya ginawa na protina W et nayarin onkiwas bilang quinone channel.
Say gap ed LH114-W loop et mangiiter na pankanawnawan nawalaan na tuloytuloy ya membrane region ed baetan na internal space na RC-LH114-W complex tan say bulk membrane (Figure 1G), imbes ya ikonekta so duaran domain panamegley na protein pore a singa ed saray protina. Say RC-LH116 complex et mipara ed sakey ya akasara ya Tch. Singa-dagum ya komplikado (22) (Figure 1H). Lapud say ikayat na quinone ed membrane et mas maples nen say ikakayat na quinone ed mainget a kanal na protina, say lukas ya LH114-W loop et makapangiter na mas maples ya RC turnover nen say akasara ya LH116 loop, tan say ikakayat na quinone ed RC et nayarin mas limitado. Pian nasubok no kasin say protina W et mangaapekta ed pananguman na quinones panamegley na RC, nanggawa kami na cytochrome oxidation assay ed sakey a konsentrasyon na ubiquinone 2 (UQ2) (sakey ya analogue na natural ya UQ10 ya mas antikey so isoprene tail to) (Figure 2E). Anggaman say kiwawala na chelated quinone et mangaamper ed suston pakadetermina na ompatnag a Michaelis constant (RC-LH114-W tan RC-LH116 et matukoy parad 0.2±0.1μM tan 0.5±0.2μM, unong ed inkauksoy da), say sankatageyan a rate na RC-LH114-W ( 4.6±0.26±0.2μM) et s-1-1-1. 28±5% ya mas baleg nen say RC-LH116 (3.6±0.1 e-RC-1 s-1).
Diad gapo et inkalkula mi ya say protina-W et walad manga 10% na core complex (16); dia, say occupancy rates na low-light, medium-light, tan high-light growth cells et 15±0.6%, 11±1% tan 0.9±0.5, % (Figure 2F). Say quantitative comparison na mass spectrometry et angipatnag ya say pangiyarum na histidine tag et ag-akabawas ed karakel na protein-W no ikompara ed wild-type strains (P = 0.59), kanian sarayan lebel et aliwan artifact na modified protein-W (Figure S10). Balet, sayan abeban okupasyon na protina-W ed RC-LH1 complex et nayarin mamaabuloy ed pigaran RC ya man-flip ed maples a peles, kanian nabawasan so mas matantan ya pansasalat na quinone/quinolone ed RC-LH116 complex. Naimano mi ya say atagey ya light occupancy rate et ag mipara ed agano nin transcriptomics data, ya mangipapanengneng ya say pufW gene expression et ondarakel no mabiskeg so liwawa (Figure S11) (23). Say pandumaan na pufW transcription tan say pangilaktip na protein-W ed RC-LH1 complex et makapawetwet tan nayarin mangipapatnag na komplikadon regulasyon na protina.
Diad RC-LH114-W, 6 cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) tan 29 β-DDM molecules so nigana tan inmodel ed satan ya 6 CDLs, 24 POPCs, 2 POPGs tan 21βDβs. RC-LH116 (Figure 5, A tan B). Diad sarayan duaran istruktura, say CDL et ngalngalin walad cytoplasmic side na complex, bangta say POPC, POPG tan β-DDM et maslak ya walad luminal side. Duaran lipid tan detergent molecules so nibiig ed αβ-1 anggad αβ-6 a rehyon na RC-LH114-W complex (Figure 5A), tan limara so nibiig ed mipara a rehyon na RC-LH116 (Figure 5B ). Mas dakel so lipid ya aromog ed biek a dapag na complex, nagkalalo la ed CDL, ya atipon ed baetan na RC tan αβ-7 ya anggad αβ-13 (Figure 5, A tan B). Saray arum ya structural resolved lipids tan detergents et walad paway na LH1 ring, tan saray well-resolved acyl chains et onnatnat ed baetan na LH1 subunits, ya temporaryon tinawag ya β-DDM ed RC-LH114-W, tan tinawag ya β-DDM ed RC A mixture na β-DDM tan PO-LH1616. Say mipadparan posisyon na chelating lipids tan detergents ed istruktura tayo et mangipapanengneng ya sikaray physiologically relevant binding sites (Figure S12A). Saray posisyon na mipara iran molekula ed Tch et walaan met na maabig a inkapara. Maamo tan Trv. Say strain 970 RC-LH1s (Figure S12, B anggad E) (9, 12) tan saray hydrogen-bonding residues na lipid head group so angipatnag na medyo maong a konserbasyon ed sequence alignment (Figure S13), ya mangipapanengneng ya say Conserved CDL ya onsisiglaot ed RC (24), sarayan pasen et nayarin napreserba ed RCH-LH1 complex.
(A tan B) Saray RC-LH114-W (A) tan RC-LH116 (B) peptides et irerepresenta na saray cartoon, tan saray pigment et irerepresenta na saray baras, ya uusaren so kolor ed Figure 1. Saray lipid et nipanengneng ed ambalanga, tan saray detergent et nipanengneng ed kolor abuek. Say UQ ya akapeket ed RC QA tan QB sites et duyaw, bangta say isolated UQ et asul. (C tan D) Pareho so panmoria ed (A) tan (B), ya anggapoy lipid ya inlaktip. (E anggad G) Pinalaknab a nengnengen na Q1(E), Q2(F) tan Q3(G) manlapud RC-LH116, a walaan na saray side chain ya mangiimpluensya ed balang sakey. Saray hydrogen bonds et nipanengneng bilang andeket ya putol-putol iran linya.
Diad RC-LH116, parehon say RC QA tan QB UQ, ya mibibiang ed panangiyalis na electron ed proseso na panagsian na karga, et nabubulok ed saray binding sites da. Balet, diad RC-LH114-W, say QB quinone et agni naresolbe tan detalyadon pantongtongan ed leksab. Nilikud ed QA tan QB quinones, duaran chelated UQ molecules (ya walad baetan na RC tan LH1 rings) so niiyan ed RC-LH114-W ya istruktura unong ed saray well-resolved head groups da (ya walad Q1 tan Q2, unong ed inkauksoy da). lugar). Pigura 5C). Duaran isoprene units so niasain ed Q1, tan say density map so manresolbe ed kompleton 10 isoprene tails na Q2. Diad istruktura na RC-LH116, taloran chelated UQ10 molecules (Q1 anggad Q3, Figure 5D) so naresolbe, tan amin ya molecules et walaan na malinew a densidad ed interon ikol (Figure 5, D anggad G). Diad duaran istruktura, say posisyon na quinone head groups na Q1 tan Q2 et walaan na ekselentin inkapara (Figure S12F), tan sikaray mibabali labat ed RC. Say Q1 et walad lolooban na W gap na RC-LH114-W (Figure 1G tan 5, C, D tan E), tan say Q2 et walad asingger na QB binding site (Figure 5, C, D) tan F). Saray napreserba ya L-Trp143 tan L-Trp269 ya residue et asingger ed Q1 tan Q2 tan mangiiter na potensyal ya π-stacking interactions (Figure 5, E tan F, tan Figure S12). Say L-Gln88, 3.0 Å manlapud distal oxygen na Q1, et mangiiter na mabiskeg ya hydrogen bond (Figure 5E); sayan residue et napreserba ed amin ya RCs likud ed sankaarawian ya relasyon (Figure S13). Say L-Ser91 et konserbatibon sinalatan na Thr ed karaklan ya arum ya RCs (Figure S13), et 3.8 Angstroms manlapud methyl oxygen na Q1, tan nayarin mangiter na makapuy iran hydrogen bonds (Figure 5E). Say Q3 et singa anggapoy espisipikon pidedeneng to, balet ta walad hydrophobic ya rehyon ed baetan na RC-M subunit tan say LH1-α subunit 5 anggad 6 (Figure 5, D tan G). Saray Q1, Q2 tan Q3 odino saray asingger ya chelated quinones et naresolbe met ed Tch. Maamo, Trv. Strain 970 tan Blc. Say istruktura na iris (9, 10, 12) et mangipapabitar ed sakey a napreserba ya auxiliary quinone binding site ed RC-LH1 complex (Figure S12G). Say limaran abulok ya UQ ed RC-LH116 et mipara ed 5.8±0.7 na kada complex ya adetermina na high performance liquid chromatography (HPLC), bangbalet say taloran abulok ya UQ ed RC-LH114-W et abeba nen say sinukat ya balor na 6.2±0.3 (Fig. S14) et mangipapanengneng ya anggapoy naresolbe ya molekula ed UQ. istruktura.
Say pseudo-symmetric L tan M polypeptides et balang sakey et walaan na limaran TMHs tan manporma na heterodimer ya mankokombina na sakey a BChl dimer, duaran BChl monomers, duaran bacteriophage (BPh) monomers, tan sakey ya aliwan Heme iron tan sakey odino duaran UQ10 molecules. Diad panamegley na kiwawala na hydrogen bonds ed terminal ketone group tan say kabkabat ya idakel to ed Rps, saray carotenoids et nilaktip ed M-subunit, ya tatawagen ya cis-3,4-dehydroorhodopin. Species (25). Say paway na membrane domain na RC-H et akaangkla ed membrane na sakey a TMH. Say inkalapagan ya istruktura na RC et mipara ed taloran subunit RC na saray mankakasakey a klase (singa say Rba). sphaeroides (PDB ID: 3I4D). Saray macrocycles na BChl tan BPh, say carotenoid backbone tan non-heme iron et mansasalet ed loob na resolution range na sarayan istruktura, ontan met ed UQ10 head group diad QA site tan say QB quinone ed RC-LH116 (Figure S15).
Say pakawalaan na duaran RC ya istruktura ya walaan na nanduruman QB site occupancy rates so mangiiter na balon pankanawnawan nausisa so agmanguman-uman iran pananguman ed konpormasyon ya kaiba na QB quinone binding. Diad RC-LH116 complex, say QB quinone et walad sigpot ya “proximal” ya posisyon (26), balet say inkaapag-apag na RC-LH114-W et anggapoy QB quinone to. Anggapoy QB quinone ed RC-LH114-W, ya makapabigla ta say complex et aktibo, mas ni nen say RC-LH116 complex ya walay structural resolved QB quinone to. Anggaman say duaran LH1 rings et man-chelate na manga anemiran quinones, limara so naresolbe ed istruktura ed sarado ya RC-LH116 ring, bangta talora labat so limitado ed istruktura ed lukas ya RC-LH114-W ring. Sayan indaak na structural disorder et nayarin mangipapatnag na mas maples ya pansalat na RC-LH114-W QB sites, mas maples ya quinone kinetics ed complex, tan mas baleg ya posibilidad ya makabaliw ed LH1 loop. Isusuheri mi ya say kakulangan na UQ ed RC QB site na RC-LH114-W et nayarin resulta na mas komplikado tan mas aktibon komplikado, tan say QB site na RC-LH114-W et tampol ya nanyelo ed UQ turnover Say espisipikon yugtan (say lolooban ed QB site et akasara) so mangipapatnag na konpormasyon na sayan aktibidad.
No anggapoy QB, say kaiba ton panag-ikot na L-Phe217 ed posisyon ya ag mipara ed UQ10 binding, lapud saya so pansengegan na spatial collision ed unonan isoprene unit na ikol (Figure 6A). Niarum ni, saray mapatnag a manunan pananguman ed konpormasyon so mapatnag, nagkalalo la ed helix de (antikey a helix ed loop ed baetan na TMH D tan E) a ditan et inyalis so L-Phe217 ed QB binding pocket tan say panag-rotate na L-Tyr223 (Figure 6A ) Pian nabuyak so hydrogen bond ed M-Ap4 framework tan isara so entrance na binding na QB pasen (Figure 6B). Say Helix de pivot ed base to, say Cα na L-Ser209 et onliliber na 0.33Å, bangta say L-Val221Cα et onliliber na 3.52Å. Anggapoy nanengneng ya pananguman ed TMH D tan E, ya nayarin ipapasen ed parehon istruktura (Figure 6A). Diad amta tayo, saya so sankaunaan ya istruktura ed natural ya RC ya mangisara ed QB site. No ikompara ed kompleton (QB-bound) ya istruktura, nanengneng ya antis ya nabawasan so quinone, kaukolan so pananguman ed itsura to pian makaloob ed quinone. Say L-Phe217 et onliliber pian manporma na π-stacking ya pidedeneng ed quinone head group, tan say helix et onalis ed paway, ya mangiter na pankanawnawa ed skeleton na L-Gly222 tan say side chain na L-Tyr223 ya manporma na hydrogen bond network ya walay stable ya hydrogen bond structure (Figure 6, A tan C).
(A) Manpapaspas ya cartoon na hologram (L chain, orange/M chain, magenta) tan apo (gray) ya istruktura, ya saray manunan residue et nipanengneng ed nengneng na singa baras ya representasyon. Say UQ10 et irerepresenta na duyaw a bar. Say tuldok-tuldok a linya et mangipapanengneng na saray hydrogen bonds ya aporma ed interon istruktura. (B tan C) Say representasyon na tapew na apolipoprotein tan say interon istruktura na singsing, ya mangipapabitar ed side chain oxygen na L-Phe217 ya asul tan L-Tyr223 ya ambalanga, unong ed inkauksoy da. Say L subunit et kolor kahel; saray M tan H subunits et anggapoy kolor da. (D tan E) Apolipoprotein (D) tan interon (E) RC QB sites [kolor na (A) unong ed inkauksoy da] tan Thermophilus thermophilus PSII (berde, asul ya walay plastic quinone; PDB ID: 3WU2) Align (58).
Ag-iilaloan, anggaman wala ray pigaran istruktura na QB-deficient RCs ya anggapoy LH1, saray pananguman ed konpormasyon ya naobserbaan ed sayan impanaral et agni nireport nensaman. Kaiba ed saraya so QB depletion structure manlapud Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) tan Rba. sphaeroides (PDB ID: 1OGV) (29), ya amin da et ngalngalin pareho ed inkalapagan ya istruktura na QB da. Diad maapit ya inspeksion ed 3PRC, anengneng ya saray molekula na LDAO (Lauryl Dimethyl Amine Oxide) ya detergent et mankakasakey ed lolooban na posisyon na QB, ya nayarin mangamper ed pangiyuksoy lamet ed sakey a saradon konpormasyon. Anggaman say LDAO et ag-onbubulok ed parehon posisyon ed 1EYS o 1OGV, sarayan RCs et imparaan ya uusaren so parehon detergent kanian nayarin pareho so epekto to. Say istruktura na kristal na Rba. Say Sphaeroides RC ya aka-crystallized ed cytochrome c2 (PDB ID: 1L9B) et singa met walaan na sarado ya QB site. Balet, diad sayan kaso, say N-terminal ya rehyon na RC-M polypeptide (ya mibabali ed QB binding site panamegley na H bond na Tyr residue ed Q helix) et walaan na aliwan natural ya konpormasyon, tan say pananguman na konpormasyon na QB et ag la nasukimat (30 ). Say makapaseguro et agtayo ni anengneng so ontan ya klase na deformation na M polypeptide ed RC-LH114-W ya istruktura, ya ngalngalin pareho ed N-terminal ya rehyon na RC-LH116 RC. Nepeg met ya imanoen ya kayari na inkaandi na detergent-based LH1 antenna, saray apolipoprotein RCs ed PDB so naresolbe, ya amawala na internal quinone pools tan lipids ed baetan na RC tan say loob na tapew na kaliberliber na LH1 ring (31, 32). Say RC et mansiansian mankikimey lapud mansiansia so amin ya cofactors, likud ed decomposable QB quinone, ya agtanton malet tan mabetbet ya naandi legan na proseso na panagparaan (33). Niarum ni, kabkabat ya say pangekal ed LH1 tan natural ya cyclic lipids manlapud RC et nayarin walay epekto to ed saray function, singa say antikey a bilay na charge-separated P+QB-state (31, 34, 35). Kanian, iisipen mi ya say kiwawala na lokal ya LH1 ring ya akapaliber ed RC et nayarin mamasiansia ed “sarado” ya QB site, diad ontan et napreserba so lokal ya kaliberliber ya asingger ed QB.
Anggaman say apolipoprotein (ya anggapoy QB quinone) tan say kompleton istruktura to et mangirerepresenta labat na duaran snapshot na turnover na QB site, imbes ya nantutumbokan ya ebento, wala ray indikasyon ya say binding et nayarin na-gate pian naprebentaan so rebinding na hydroquinone Pian naprebentaan so substrate inhibition. Say pidedeneng na quinolol tan quinone ya asingger ed QB site na apolipoprotein et nayarin mandumaan, ya pansengegan na pangipulisay ed satan na RC. Abayag lan iproproponin say pananguman ed konpormasyon et walay papel to ed panag-apiger tan panagpaabeba na saray quinones. Say abilidad na saray frozen RCs ya mamawas ed quinones kayari na dark adaptation et aderal (36); Ipapanengneng na X-ray crystallography ya sayan destroso et lapud saray QB quinones ya aka-trap ed “distal” ya konpormasyon ya manga 4.5 Å manlapud aktibon proximal ya posisyon (26) , 37). Isusuheri mi ya sayan distal binding conformation et sakey a snapshot na intermediate state ed baetan na apolipoprotein tan say full ring structure, ya ontumbok ed inmunan pidedeneng ed quinone tan say pangilukas na QB site.
Say type II RC ya naromog ed PSII complex na pigaran phototrophic bacteria tan cyanobacteria, algae tan tanaman et walaan na structural tan functional conservation (38). Say structural alignment ya nanengneng ed Figure 6 (D tan E) et mangipapabitar na pampareho na PSII RCs tan say QB site na bacterial RC complex. Sayan panangikompara et abayag lan modelo parad panaral ed saray maapit a sistema na quinone binding tan reduction. Insuheri na saray akaunan publikasyon ya saray pananguman ed konpormasyon et kaiba na PSII ya ibawas na quinones (39, 40). Kanian, no ikonsidera so ebolusyonaryon panangipreserba na RC, sayan agni naobserbaan ya mekanismo na panag-apiger et nayarin onaplika met ed QB site na PSII RC ed saray oxygenated phototrophic plants.
Rps ΔpufW (unlabeled pufW deletion) tan PufW-His (C-terminal 10x His-tagged protein-W ya nanlapud natural ya pufW locus) ya strain. say palustris CGA009 et adeskribe ed akaunan kimey mi (16). Sarayan strain tan say isogenic wild-type parent et naala ed freezer diad panamegley na pangi-streak na daiset a bilang na selula ed PYE (kada sakey 5 g litro -1) (ya insimpen ed LB ed -80 °C, ya walay 50% ( w/v) glycerol) ya protina, yeast extract tan succinate) agar [1.5%) (w/v) plate. Say plato et in-incubate ed sanlabi ed ambilunget diad temperatura na kuarto diad silong na anaerobic iran kondisyon, insan inliwawaan na amputin liwawa (~50 μmolm-2 s-1) ya inter na OSRAM 116-W halogen bulbs (RS Components, UK) ed loob na 3 anggad 5 ya agew anggad pinmatnag so sakey a kolonya. Sakey a kolonya so inusar pian na-inoculate so 10 ml na M22+ medium (41) ya inaruman na 0.1% (w/v) casamino acids (ya tatawagen ya M22). Say kultura et pinabaleg ed silong na abeban kondisyon na oxygen diad ambilunget ed 34°C ya inyugyog ed 180 rpm ed loob na 48 oras, insan 70 ml na kultura so ininoculate ed parehon kondisyon ed loob na 24 oras. Say semi-aerobic culture ya walay volume ton 1 ml et inusar pian na-inoculate so 30 ml na M22 medium ed 30 ml ya universal screw-top transparent glass bottle tan i-irradiate na agitation (~50μmolm-2 s-1) ed loob na 48 oras panamegley na sterile magnetic force Stirring rod. Insan 30 ml na kultura so ininokulaan na manga 1 litro na kultura diad parehon kondisyon, ya inusar pian ibakunaan so manga 9 litro na kultura ya insilew ed ~200 μmolm-2 s-1 ed loob na 72 oras. Saray selula et inani panamegley na centrifugation ed 7132 RCF ed loob na 30 minutos, insuspende lamet ed ~10 ml na 20 mM tris-HCl (pH 8.0), tan insimpen ed -20°C anggad nakaukolan.
Kayari na panagtunaw, idagdag so pigaran kristal na deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) tan duaran Roche holoenzyme protease inhibitor tablets (Merck, UK) ed saray resuspended ya selula. Diad 20,000 psi French pressure cell (Aminco, USA), saray selula et aderal na 8 anggad 12 danay. Kayari na pangekal ed saray ag nabuyak ya selula tan saray ag natunaw ya debris diad panamegley na centrifugation ed 18,500 RCF ed loob na 15 minutos ed 4°C, say membrane et inprecipitate manlapud pigmented lysate diad panamegley na centrifugation ed 113,000 RCF ed loob na 2 oras ed 43,000°C. Ibantak so soluble fraction tan i-resuspend so kolor na membrane ed 100 anggad 200 ml na 20 mM tris-HCl (pH 8.0) tan i-homogenize anggad anggapo lay nanengneng ya aggregates. Say akabitin ya membrane et in-incubate ed 20 mM tris-HCl (pH 8.0) (Anatrace, USA) ya walay 2% (w/v) β-DDM ed loob na 1 oras ed ambilunget ed 4°C ya walay maalwar ya pangigalaw. Insan i-centrifuge ed 70°C pian natunaw so 150,000 RCF ed 4°C ed loob na 1 oras pian naekal iray nakekeraan ya ag natunaw.
Say solubilizing membrane manlapud ΔpufW strain et inyaplika ed 50 ml DEAE Sepharose ion exchange column ya walay taloran column volumes (CV) na binding buffer [20 mM tris-HCl (pH 8.0) ya walay 0.03% (w / v) β-DDM]. Urasan so kolum na duaran CV binding buffer, insan urasan so kolum na duaran binding buffer ya walay 50 mM NaCl. Say RC-LH116 complex et in-elute ed linear gradient ya 150 anggad 300 mM NaCl (diad binding buffer) ed 1.75 CV, tan say nakekeraan ya binding complex et in-elute ed binding buffer ya walay 300 mM NaCl ed 0.5 CV. Kolaen so absorption spectrum ed baetan na 250 tan 1000 nm, pansiansiaen so fraction ya walay absorbance ratio (A880/A280) ya mas atagey nen say 1 diad 880 anggad 280 nm, lawen itan na mamidua ed binding buffer, tan usaren lamet so parehon paraan ed DEAE column On purification. I-dilute iray fraction ya walay A880/A280 ratios ya mas atagey nen say 1.7 tan A880/A805 ratios ya mas atagey nen say 3.0, gawaen so komatlon round na ion exchange, tan pansiansiaen iray fractions ya walay A880/A280 ratios ya mas atagey nen say 2.2 tan A880/A805 ratios ya mas atagey nen say 0.0.0. Say partially purified complex et inkonsentra ed ~2 ml ed sakey ya Amicon 100,000 molecular weight cut-off (MWCO) centrifugal filter (Merck, UK), tan inkarga ed sakey ya Superdex 200 16/600 size exclusion column (GE Healthcare, US) ya walay 200 mM NaCl buffer, insan in-elution ed parehon buffer. CV. Kolektaen iray absorption spectra na size exclusion fraction, tan ikonsentray saray absorption spectra ya walay A880/A280 ratios ed 2.4 tan A880/A805 ratios ed 5.8 anggad 100 A880, tan usaren iratan a tampol parad panagparaan na cryo-TEM grid odino panag-imbantak Pansiansiaen ed -80°C anggad nakaukolan.
Say solubilizing membrane manlapud PufW-His strain et inyan ed 20 ml ya HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) ya walay 200 mM NaCl tan 0.03% (w/w)) ed IMAC buffer ( GE Healthcare). v) β-DDM]. Say kolum et inurasan na limaran CV na IMAC buffer, insan na limaran CV na IMAC buffer ya walay 10 mM histidine. Say core complex et inekal ed kolum ya walay limaran IMAC buffers ya walay 100 mM histidine. Say fraction ya walaan na RC-LH114-W complex et ikonsentray anggad ~10 ml diad sakey a stirred tank ya walay Amicon 100,000 MWCO filter (Merck, UK), ya inlaok na 20 beses na binding buffer, insan inyarum ed 25 ml Diad DEAE Sepharose column, apatiran CV buffer so inusar ya akauna ed buffer. Urasan so kolum na apatiran CV binding buffer, insan i-elute so complex ed waloran CV diad linear gradient ya 0 anggad 100 mM NaCl (diad binding buffer), tan say nakekeraan ya apatiran CV ya walaan na 100 mM binding buffer Saray nakekeraan ya complex ya in-elute ed sodium chloride ya inlaok ed A888/2008 ratio et mas atagey nen say 2004. Say A880/A805 ratio ya mas atagey nen say 4.6 fractions et inkonsentra ed ~2 ml ed sakey ya Amicon 100,000 MWCO centrifugal filter, tan pinano na 1.5 CV IMAC ya masakbay. Kolektaen so absorption spectra na saray size-exclusion fractions tan ikonsentray absorption spectra ya walay A880/A280 ratios ed 2.1 tan A880/A805 ratios ed 4.6 anggad 100 A880, ya tampol ya usaren parad panagparaan na frozen TEM grid odino isimpen ed -80°C anggad nakaukolan.
Say Leica EM GP immersion freezer so inusar pian iparaan iray low temperature TEM grids. Say complex et inlaok ed IMAC buffer ya anggad A880 na 50, insan 5μl so inkarga ed balon glow-discharged QUANTIFOIL 1.2/1.3 carbon-coated copper mesh (Agar Scientific, UK). I-incubate so grid ed 20°C tan 60% ya relatibon humidity ed loob na 30 s, insan i-blot ya pamaraan ed loob na 3 s, insan i-quench ed liquid ethane ed -176°C.
Say data na RC-LH114-W complex et nirekord ed eBIC (Electronic Bioimaging Center) (British Diamond Light Source) ya walay Titan Krios microscope, ya mankikimey ed maples ya boltahe ya 300kV, ya walay nominal ya magnification ya 130,000× tan enerhiya na Choo-se ya 2000V. Say Gatan 968 GIF Quantum ya walay K2 peak detector so inusar pian irekord iray imahen ed counting mode pian makakolekta na data. Say na-calibrate ya kabaleg na pixel et 1.048Å, tan say dose rate et 3.83 e-Å-2s-1. Naala so pelikula ed loob na 11 segundo tan inapag ed 40 a parte. Usaren so carbon-coated area pian na-refocus so mikroskopyo, insan mankolekta na taloran pelikula ed kada abut. Diad dagup, 3130 ya pelikula so nakolekta, ya say defocus values ​​et walad baetan na -1 anggad -3μm.
Say datos parad RC-LH116 complex et naala ya inusar so parehon mikroskopyo diad Asterbury Biostructure Laboratory (University of Leeds, UK). Say datos et naala ed counting mode ya walay magnification ya 130 k, tan say kabaleg na pixel et in-calibrate ed 1.065 Å ya walay dosis ya 4.6 e-Å-2s-1. Say pelikula et nirekord ed loob na 12 segundo tan inapag ed 48 a parte. Diad dagup, 3359 ya pelikula so nakolekta, ya say defocus values ​​et walad baetan na -1 anggad -3μm.
Amin ya panagproseso na data et gagawaen ed Relion 3.0 pipeline (42). Usaren so Motioncorr 2 (43) pian ipetek so galaw na sinag panamegley na dose weighting, insan usaren so CTFFIND 4.1 (44) pian naamtaan so CTF (contrast transfer function) ya parameter. Saray kaslakan ya photomicrographs kayari na sarayan inmunan yugtan na panagproseso et nanengneng ed Figure 2. S16. Say automatic selection template et nagawa diad manual ya panagpili na manga 250 pixels na 1000 particles ed 250-pixel frame tan anggapoy reperensya ya two-dimensional (2D) ya klasipikasyon, kanian ag-ipulisay iramay klasipikasyon ya makaabot ed kontaminasyon na sample odino anggapoy naimano iran katangian. Insan, ginawa so automatikon panagpili ed amin ya microphotographs, tan say RC-LH114-W et 849,359 a partikulo, tan say RC-LH116 complex et 476,547 a partikulo. Amin ya pinilin partikulo et dinmalan ed duaran round na non-reference 2D classification, tan kayari na kada run, saray partikulo ya akasabi ed carbon area, sample contamination, anggapoy mapatnag ya features odino mabiskeg ya mansasalet ya particles et ag-inawat, kanian 772,033 (90.9%) tan 359,678 (75.5) particles so inusar parad klasipikasyon na 3D 3D. RC-LH114-W tan RC-LH116 unong ed inkauksoy da. Say inmunan 3D reference model et ginawa ya inusar so stochastic gradient descent method. Diad pangusar ed inmunan modelo bilang reperensya, saray pinilin partikulo et niklasipika ed apatiran kategorya diad 3D. Diad pangusar ed modelo ed sayan kategorya bilang reperensya, manggawa na 3D refining ed saray partikulo ed sankabalgan a kategorya, insan usaren so inmunan 15Å low-pass filter pian sakbongan so solvent area, mangiyan na 6 pixels na soft edges, tan i-post-process iray pixel pian ipetek so Gatan K2 peak Modulation transfer function na top detector. Parad RC-LH114-W dataset, sayan inmunan modelo et inuman diad pangekal ed mabiskeg a densidad ed gilig na maskara (ya ag-akakonekta ed core complex density ed UCSF Chimera). Saray resulta na modelo (saray resolusyon na RC-LH114-W tan RC-LH116 et 3.91 tan 4.16 Å, unong ed inkauksoy da) so inusar bilang reperensya parad komaduan round na 3D classification. Saray partikulo ya inusar et agrupo ed inmunan 3D ya klase tan anggapoy mabiskeg a siglaotan da ed kaliberliber. Manpapaspas odino anggapoy mapatnag iran itsura na istruktura. Kayari na komaduan round na 3D classification, say kategorya ya walaan na sankatageyan ya resolusyon so pinili [Parad RC-LH114-W, sakey a kategorya et 377,703 particles (44.5%), parad RC-LH116, walay duaran kategorya, ya mankantidad na 260,752 particles (54.7% W%), no pareho labat so inlinya ra kayari na saraya. inmunan panag-ikot ya walay melag a pandumaan]. Saray pinilin partikulo et naala lamet ed 400-pixel ya kahon tan napino na 3D. Say solvent mask et ginawa ya uusaren so inmunan 15Å low-pass filter, 3 pixel map expansion tan 3 pixel soft mask. Diad pangusar na per-particle CTF refinement, per-particle motion correction tan say komaduan round na per-particle CTF refinement, say 3D refinement, solvent masking tan post-processing so gagawaen kayari na kada kundang pian lalon napino so resulta na texture. Diad pangusar ed FSC (Fourier Shell Correlation Coefficient) cut-off value ya 0.143, say resolusyon na pinal iran modelo na RC-LH114-W tan RC-LH116 et 2.65 tan 2.80Å, unong ed inkauksoy da. Say FSC curve na unor a modelo et nanengneng ed Figure 2. S17.
Amin ya protein sequences et na-download manlapud UniProtKB: LH1-β (PufB; UniProt ID: Q6N9L5); LH1-α (PufA; UniProtID: Q6N9L4); RC-L (PufL; UniProt ID: O83005); RC-M (PufM; UniProt ID: A0A4Z7); RC-H (PuhA; UniProt ID: A0A4Z9); Protein-W (PufW; UniProt ID: Q6N1K3). Say SWISS-MODEL (45) so inusar pian manggawa na homology model na RC, ya walaan na saray protina ya nantutumbokan na RC-L, RC-M tan RC-H tan say istruktura na kristal na Rba. sphaeroides RC so inusar bilang template (PDB ID: 5LSE) (46). Usaren so “fit map” tool ed UCSF Chimera pian na-fit so ginawan modelo ed mapa (47), napaaligwas so istruktura na protina, tan cofactor [4×BChl a (monomer library residue name = BCL), 2×BPh a (BPH), sakey odino duaran klase na UQ10 (U10), sakey ya non-heme iron (Fe) tan 34 hexadichocarycoline (QAK)] usaren so Coot (48) pian mangarum. Lapud anggapoy QAK ed monomer library, in-parameterize itan ya inusar so eLBOW tool ed PHENIX (49).
Insan, say LH1 subunit so ginawa. Diad gapo, say automatic construction tool ed PHENIX (49) so inusar pian automatikon nagawa so kabiangan na LH1 sequence ya inusar so mapa tan say LH1-α tan LH1-β protein sequences bilang input. Pilien so sankakompletoan ya LH1 subunit, i-extract tan i-load ed Coot, manual ya idagdag so kulang ya sequence ed satan, tan manual ya i-refine so interon istruktura antis ya idagdag so duaran BCls a (BCL) tan sakey a spirilloxanthin (CRT) [unong ed matukoy ya Rps Say densidad na LH1 complex tan say kabkabat ya carotenoid content. Species (17)]. Kopyaen so kompleton LH1 subunit, tan usaren so UCSF Chimera “Docking Map Tool” pian maka-dock ed asingger ya aliwan modelo ya lugar na LH1 density, insan i-refine ed Coot; uliten so proseso anggad amin ya LH1 subunits et namodelo la. Parad RC-LH114-W ya istruktura, diad pangala ed ag-in-alocate ya densidad ed Coot, say protina et naapag manlapud saray nakekeraan ya aliwan protina ya kabiangan ed USCF Chimera map tan say Autobuild tool et nausar pian niletneg so inmunan modelo, tan saray nakekeraan ya subunits (protein-W) Modeling. Diad PHENIX (49). Ilaktip so antokaman ya kulang ya sequence ed resulta na modelo ed Coot (48), insan manual ya i-refine so interon subunit. Say nakekeraan ya ag niiter ya densidad et onkana ed kombinasyon na lipids (PDB monomer library ID na CDL = CDL, POPC = 6PL tan POPG = PGT), β-DDM detergent (LMT) tan UQ10 molecules (U10). Usaren so PHENIX optimization (49) tan manual optimization ed Coot (48) pian naperpekto so kompleton inmunan modelo anggad ag la napaaligwas so model statistics tan say visual quality na fit. Kaunoran, usaren so LocScale (50) pian napaatagey so lokal a mapa, insan manggawa na pigaran arum nin siklo na panagmodelo ed ag-inalokar a densidad tan automatiko tan manual ya panag-optimize.
Saray nanduruman peptide, cofactor tan arum niran lipid tan quinones ya akapeket ed loob na nanduruman densidad da et nanengneng ed Figures 1 tan 2. S18 anggad S23. Say estadistikal ya impormasyon na unor a modelo et nanengneng ed Table S1.
Likud no walay arum ya nibaga, say UV/Vis/NIR absorption spectra et naala ed Cary60 spectrophotometer (Agilent, USA) diad 1 nm ya interval manlapud 250 nm anggad 1000 nm tan say integration time et 0.1s.
I-dilute so sample ed quartz cuvette ya walay 2 mm ya dalan ya anggad A880 na 1, tan kolektaen so absorption spectrum ed baetan na 400 anggad 1000 nm. Saray pabilog ya dichroic spectra et naala ed Jasco 810 spectropolarimeter (Jasco, Japan) ed 1 nm ya interval ed baetan na 400 nm tan 950 nm diad scan rate ya 20 nm min-1.
Say molar extinction coefficient et nadetermina diad pangi-dilute ed core complex ed A880 ya manga 50. I-dilute so 10μl ya volume ed 990μl binding buffer o methanol, tan kolektaen ya tampol so absorption spectrum pian nabawasan so BChl degradation. Say BChl ya karga na kada sample na methanol et kinalkula panamegley na extinction coefficient ed 771 nm na 54.8 mM-1 cm-1, tan say extinction coefficient et nadetermina (51). Inapag so asukat ya konsentrasyon na BChl ed 32 (RC-LH114-W) odino 36 (RC-LH116) pian naamtaan so konsentrasyon na core complex, ya usaren pian naamtaan so absorption spectrum na parehon sample ya naala ed buffer Extinction coefficient. mipara. Taloran paulit-ulit ya sukat so ginawa parad kada sample, tan say average ya absorbance na BChl Qy maximum so inusar parad kalkulasyon. Say extinction coefficient na RC-LH114-W ya nasukat ed 878 nm et 3280±140 mM-1 cm-1, bangta say extinction coefficient na RC-LH116 ya nasukat ed 880 nm et 3800±30 mM-1 cm-1.
Say UQ10 et nabilang unong ed paraan ed (52). Diad antikey, say reverse phase HPLC (RP-HPLC) et ginawa ya inusar so Agilent 1200 HPLC system. Itunaw so manga 0.02 nmol na RC-LH116 o RC-LH114-W ed 50μl na 50:50 methanol:chloroform ya walay 0.02% (w/v) ferric chloride, insan i-inject so pre-equilibrated Beckman Coulter Ultrasphere ODS 46 mml ed 1-1-1-m. 40°C ed HPLC solvent (80:20 methanol:2-propanol) ed ×25 cm ya kolum. Manggawa na isocratic elution ed HPLC solvent pian nabantayan so absorbance ed 275 nm (UQ10), 450 nm (carotenoids) tan 780 nm (BChl) ed loob na 1 oras. Say peak ed 275 nm chromatogram ed 25.5 minutos et intipon, ya anggapoy arum ya nanengneng ya compound. Say integrated area et uusaren pian kalkulaen so molar ya karakel na UQ10 ya naala base ed calibration curve ya kinalkula manlapud pangi-injection na puron estandarte manlapud 0 anggad 5.8 nmol (Figure S14). Kada sample et inanalisa ed taloran replika, tan say nireport ya error et mipara ed SD na average.
Say solusyon ya walay RC-LH1 complex ya walay sankatageyan ya Qy absorption ya 0.1 et imparaan ya walay 30 μM ya abawasan ya horse heart cytochrome c2 (Merck, UK) tan 0 anggad 50 μMUQ2 (Merck, UK). Taloran 1-ml ya sample so imparaan ed kada konsentrasyon na UQ2 tan in-incubate ed sanlabi ed ambilunget ed 4°C pian naseguro so sigpot ya pakaadapta ed ambilunget antis ya sukaten. Say solusyon et inkarga ed OLIS RSM1000 modular spectrophotometer ya walay 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm middle tan 0.6 mm outlet slits. Say 600 nm ya andukey a pass filter so inyan ed lolooban na sample phototube tan say reference photomultiplier tube pian ag-onloob so excitation light. Say absorbance et nabantayan ed 550 nm ya walay integration time ya 0.15 s. Say excitation light et ompaway ed 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) panamegley na fiber optic cable ya 90% so intensidad to panamegley na DC2200 controller (Thorlabs Ltd., UK) tan ompaway ed lapuan na liwawa diad anggulo ya 90°. Say panagsukat a sinag et akaarap ed espiho pian ipawil so antokaman a liwawa ya ag inmuna ya nasepsep na sample. Bantayan so absorbance 10 s antis na 50 s ya liwawa. Insan say absorbance et lalon inbantayan ed loob na 60 s ed ambilunget pian nanengneng no panon karakel so spontaneous ya pambawas na quinolol ed cytochrome c23 + (nengnengen so Figure S8 parad raw data).
Say datos et inproseso diad pangi-fit na linear initial rate ed loob na 0.5 anggad 10 s (depende ed konsentrasyon na UQ2) tan in-average so rate na amin ya taloran sample ed kada konsentrasyon na UQ2. Say RC-LH1 ya konsentrasyon ya kinalkula na nanduruman extinction coefficient et inusar pian nauman so rate ed catalytic efficiency, ya in-plot ed Origin Pro 2019 (OriginLab, USA), tan in-fit ed Michaelis-Menten model pian naamtaan so mapatnag ya Km tan Kcat values.
Parad transient absorption measurements, say RC-LH1 sample et inlaok ed ~2μM ed IMAC buffer ya walay 50 mM sodium ascorbate (Merck, USA) tan 0.4 mM Terbutin (Merck, USA). Say ascorbic acid et uusaren bilang sacrificial electron donor, tan say tert-butaclofen et uusaren bilang QB inhibitor pian naseguro ya say manunan RC donor et mansiansian nabawasan (salanti, aliwan photooxidized) legan na proseso na panagsukat. Manga 3 ml na sample so iyarum ed sakey a custom rotating cell (manga 0.1 m so diametro to, 350 RPM) ya walay 2 mm ya karukey na optical path pian naseguro ya say sample ed laser path et walaan na magenap a panaon parad dark adaptation ed baetan na saray excitation pulses. Mangusar na ~100-fs laser pulses pian napalaknab so Ti: Sapphire laser system (Spectra Physics, USA) pian na-excite so sample ed 880 nm diad repetition rate ya 1 kHz (20 nJ parad NIR o 100 nJ parad Vis). Antis ya mankolekta na data, ipaarap so sample ed excitation light ed loob na manga 30 minutos. Say pakapatnagan et pansengegan na ag-inkayadyari na QA (posiblin mamawas na QA na maminsan odino mamidua). Balet tandaan ya sayan proseso et nauman ta kayari na andukey a panaon na dark adaptation, say RC et kalkalnan ompawil ed QA ya aktibidad. Say Helios spectrometer (Ultrafast Systems, USA) so inusar pian sukaten iray transient spectra ya walay delay time ya -10 anggad 7000 ps. Usaren so Surface Xplorer software (Ultrafast Systems, USA) pian na-ungroup iray data sets, insan i-merge tan i-standardize. Usaren so CarpetView software package (Light Conversion Ltd., Lithuania) pian nausar so nankakasakey ya data set pian makala na differential spectra ya misiglaotan ed decay, odino mangusar na function ya manlalaok na dakel ya exponents ya walay instrument response pian onkana ed single-wavelength spectral evolution ed Origin (OriginLab, USA).
Unong ed asalambit ed tagey (53), sakey a photosynthetic film ya walay LH1 complex ya anggapoy RC tan peripheral LH2 antenna so imparaan. Say membrane et inlaok ed 20 mM tris (pH 8.0) insan inkarga ed quartz cuvette ya walay 2 mm ya optical path. Say 30nJ laser pulse so inusar pian na-excite so sample ed 540 nm ya walay delay time ya -10 anggad 7000 ps. Iproseso so data set unong ed nideskribe parad Rps. pal sample.
Say membrane et in-pellet panamegley na centrifugation ed 150,000 RCF ed loob na 2 oras ed 4°C, insan say absorbance to ed 880 nm et in-resuspended ed 20 mM tris-HCl (pH 8.0) tan 200 mM NaCl. Tunaw so membrane diad kalkalnan pangigalaw ed 2% (w/v) β-DDM ed loob na 1 oras diad ambilunget ed 4°C. Say sample et inlaok ed 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) ya anggad konsentrasyon na protina ya 2.5 mg ml-1 (Bio-Rad analysis). Say kaaruman ya proseso so ginawa manlapud akaunan nipalapag a paraan (54), ya ginmapo ed impanlaok na 50 μg a protina ed dagup na 50 μl TEAB ya walaan na 1% (w/v) sodium laurate (Merck, UK). Kayari na sonication ed loob na 60 s, binawasan itan na 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) ed 37°C ed loob na 30 minutos. Parad S-alkylation, i-incubate so sample ed 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) tan idagdag itan manlapud 200 mM isopropanol stock solution ed loob na 10 minutos ed temperatura na kuarto. Say proteolytic digestion et ginawa diad pangiyarum na 2 μg trypsin/endoproteinase Lys-C mixture (Promega UK) tan in-incubate ed 37°C ed loob na 3 oras. Say laurate surfactant et inala diad pangiyarum na 50 μl ethyl acetate tan 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) tan in-vortexing ed loob na 60 s. Say inkaapag-apag na phase et insulong na centrifugation ed 15,700 RCF ed loob na 5 minuto. Unong ed protocol na manggagawa, sakey a C18 spin column (Thermo Fisher Scientific, UK) so inusar pian maalwar ya na-aspirate tan na-desalt so abeban parte ya walaan na peptide. Kayari na impamamaga panamegley na vacuum centrifugation, say sample et tinunaw ed 0.5% TFA tan 3% acetonitrile, tan 500 ng so inanalisa panamegley na nanoflow RP chromatography ya inlaok ed mass spectrometry ya inusar iray parametro na sistema ya indetalye la.
Usaren so MaxQuant v.1.5.3.30 (56) parad pakabidbiran tan bilang na protina pian anapen so Rps. palustris proteome database (www.uniprot.org/proteomes/UP000001426). Say mass spectrometry proteomics data et indeposito ed ProteomeXchange Alliance panamegley na PRIDE partner repository (http://proteomecentral.proteomexchange.org) diad silong na dataset identifier PXD020402.
Parad panagsukimat panamegley na RPLC ya inlaok ed electrospray ionization mass spectrometry, say RC-LH1 complex et imparaan manlapud wild-type Rps. Diad pangusar ed akaunan nipalapag a paraan (16), say konsentrasyon na protina ya napapawala ed saray selula na palustris et 2 mg ml-1 ed 20 mM Hepes (pH 7.8), 100 mM NaCl tan 0.03% (w/v) β- (Bio-Rad analysis) ) DDM. Unong ed protocol na manggagawa, mangusar na 2D purification kit (GE Healthcare, USA) pian naala so 10 μg ya protina panamegley na precipitation method, tan tunawen so precipitate ed 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile tan 20% (v/v) danum. Limaran microliters so inaral panamegley na RPLC (Dionex RSLC) ya inlaok ed mass spectrometry (Maxis UHR-TOF, Bruker). Usaren so MabPac 1.2×100 mm column (Thermo Fisher Scientific, UK) parad panangibiig ed 60°C tan 100μlmin -1, ya walay gradient ya 85% (v / v) solvent A [0.1% (v / v) FA tan 0.02% (V/v) TFA aqueous solution] ed solvent(v / v) [0.1%(v/v) FA tan 0.02%(v/v) ed 90%(v/v) acetonitrile TFA] Diad pangusar na estandarten electrospray ionization source tan default parameters ed loob na masulok ya 60 minutos, say mass spectrometer et makagamor na 100 anggad 2750 m/z (mass to-charge ratio). Diad tulong na ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), i-map so mass spectrum ed saray subunits na complex.
Saray selula so pinabaleg ed loob na 72 oras diad silong na 100 ml NF-low (10μMm-2 s-1), medium (30μMm-2 s-1) odino high (300μMm-2 s-1) ya liwawa. M22 medium (M22 medium ya anggapoy ammonium sulfate tan say sodium succinate et sinalatan na sodium acetate) diad 100 ml ya screw-top bottle (23). Diad limaran 30-s ya siklo, 0.1 micron ya glass beads so in-bead ed volume ratio ya 1:1 pian na-lyse iray selula tan in-chill ed yelo ed loob na 5 minutos. Say ag natunaw a bengatla, saray ag nabuyak ya selula tan saray glass beads et inekal panamegley na centrifugation ed 16,000 RCF ed loob na 10 minutos diad benchtop microcentrifuge. Say membrane et insian ed Ti 70.1 rotor ya walay 100,000 RCF ed 20 mM tris-HCl (pH 8.0) ya walay 40/15% (w/w) ya sucrose gradient ed loob na 10 oras.
Unong ed adeskribe ed akaunan kimey tayo, say immunodetection na His tag ed PufW (16). Diad antikey, say purified core complex (11.8 nM) odino say membrane ya walaan na parehon konsentrasyon na RC (ya nadetermina panamegley na oxidation ya ibawas so abawasan ya pandumaan na spectrum tan itutunos so karga ed mantsa na gel) ed 2x SDS loading buffer (Merck, UK) ya inlaok na mamidua. Saray protina et insian ed replica 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). Sakey a gel so tinina na Coomassie Brilliant Blue (Bio-Rad, UK) pian na-load tan nanengneng so RC-L subunit. Say protina ed komaduan gel et inyalis ed methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) parad immunoassay. Say PVDF membrane et in-block ed 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 tan 5% (w / v) ya skimmed milk powder, insan in-incubate ed anti-His primary antibody (diad Dilute the antibody buffer [50 mM H-HCl) 7.6), 150 mM NaCl tan 0.05% (v/v) Tween-20] ed 1:1000 A190-114A, Bethyl Laboratories, USA) ed loob na 4 oras. Kayari na 3 beses ya impanuras ed loob na 5 minuto ed antibody buffer, say membrane et inlaok ed horseradish peroxidase (Sigma-Aldrich, UK) ya anti-mouse secondary antibody (inlaok na 1:10,000 ed antibody buffer) I-incubate pian nanengneng (5 minuto kayari na 3 ya inuras ed antibody buffer) ya uusaren so WAR EST 2000. chemiluminescence substrate (Cyanagen, Italy) tan Amersham Imager 600 (GE Healthcare, UK).
Diad pangidrowing na intensity distribution na kada stained gel odino immunoassay lane, ya itipon so lugar ed silong na peak tan kalkulaen so intensity ratio na RC-L (stained gel) tan Protein-W (immunoassay), diad ImageJ (57) Iproseso so imahen. Sarayan ratio et inuman ed molar ratios diad pangipasen ya say ratio na RC-L ed protein-W ed puron RC-LH114-W sample et 1:1 tan in-normalize so interon data set unong ed satan.
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David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe, Mark J. Dickman, Dewey Holten, Christine Kirma, Hicoil Necoil, Neilcoil.
Say high-resolution ya istruktura na light trap 1 complex ed reaction center et mangiiter na balon pakatalos ed quinone dynamics.
David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe, Mark J. Dickman, Dewey Holten, Christine Kirma, Hicoil Necoil, Neilcoil.
Say high-resolution ya istruktura na light trap 1 complex ed reaction center et mangiiter na balon pakatalos ed quinone dynamics.
©2021 American Association for the Advancement of Science. akareserba so amin a kanepegan. Say AAAS et partner na HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef tan COUNTER. ScienceAdvances ISSN 2375-2548.