I kare ni†Kabedo me kare ni: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Kabedo ma ki lwongo ni reaction center light-harvesting complex 1 (RC-LH1) tye gin ma pire tek ma kwako ler ma ki lwongo ni purple phototrophic bacteria. Wa kelo jami aryo ma ki lwongo ni cryo-electron microscopy ma ki lwongo ni RC-LH1 complex ma aa ki i Rhodopseudomonas palustris. Kit ma 2.65-Å ma ki yubu kwede RC-LH114-W tye ki 14 subunit LH1 ma orumo RC, ma ki turo ki protein W, kun complex ma pe ki protein-W tye ki RC ma orumo RC. Ki umu 16 subunit LH1 loop. Apoka poka pa jami eni miyo ngec ikom kit ma quinone tiyo kwede i RC-LH1 complex, medo ki alokaloka ma onongo peya ki ngeyo i kare ma ki keto quinone i RC QB site, kacel ki kabedo pa auxiliary quinone binding sites, ma konyo me tero gi i RC. Kit ma W protein ni tye kwede ma pat pat gengo cego pa LH1 loop, ma miyo yoo me weko quinone/quinolone gi loko oyoto.
Teko ma aa ki i kom jami makwo ma ki lwongo ni photosynthesis twero gwoko kwo pa dano weny i lobo, ki tye ki kero madit me tic ki jami ma kwo ma aa ki i ceng. I kare ma tye ka kelo yub me dongo lobo, kwidi ma nen calo ler ma nen calo ler bene nyutu teko ma pat pat ki kero me tic ki kom. Gitwero gengo tic ki lero ki dongo calo kwidi ma kwo i piny macol, gitwero yubu nitrogen ki carbon dioxide, gi yubu hydrogen, ki balo jami ma ngwece kur (1-3). Me miyo teko bot jami eni, lero omyera ki lok oyot ki maber me doko teko ma ki yubu ki jami ma ki lwongo ni chemical. Tic man cako ka gin ma mako lero ni mako lero ki tero teko ma ki mako ni i kabedo ma ki lwongo ni reaction center (RC), ki dong cako poko kin mac (4 – 7). Gin ma pire tek me yubu lero i kom kwidi ma ryangi ryangi ryangi tye ki dul 2 RC, ma ki guro ki light-harvesting complex 1 (LH1), ma yubu RC-LH1 core complex. LH1 ki yubu ki dul ma ki lwongo ni curved αβ heterodimers, ma acel acel mako jami aryo ma ki lwongo ni bacterial chlorophyll (BChl) a molecules ki acel onyo aryo carotenoids (8-12). LH1 antenna ma yot loyo tye ki 16 onyo 17 αβ heterodimers ma guro RC (9-13) i dul ma ki umu, ento i core complexes mukene, transmembrane peptides gi turo rwom me LH1 ma orumo, ma weko quinol/quinone diffusion i kin RC ki cytochrome bc complex (1111, 13). Gin ma ki lwongo ni purple phototrophic plant Rhodopseudomonas (Rps.) tye gin makwo ma twero niang teko ki electron ma konyo dongo pa ceng. Kit ma ki yubu kwede gin ma ki lwongo ni Rps. Kit ma ki yubu kwede palustris RC-LH1 ni tye RC, ma ki guro ki 15 heterodimeric LH1 loops, ma ki turo ki gin ma pe ki ngeyo ma ki lwongo ni "Protein W" (14). Protein-W ki ngeyo ni RPA4402, ma tye 10.5kDa protein ma pe ki ngeyo ki transmembrane helices adek (TMH) (16). Wa tye ka tamo me loko nying rpa4402 gene ma coyo protein W me doko pufW wek obed ma rwate ki nying ma kitiyo kwede pi gene ma coyo RC-L, M (pufL, pufM) ki LH1α, β (pufA, pufB) subunits. Ma omyera, protein-W tye i 10% pa RC-LH1, ma nyutu Rps. palustris kelo jami aryo ma pat pat ma ki lwongo ni RC-LH1. Kany, wa tito jami ma ki lwongo ni cryo-EM (cryo-EM) ma tye ki rwom ma malo pa jami aryo ma pire tek, acel ki protein W ki 14 αβ heterodimers, mukene ma pe ki protein W ki 16 Heterodimer LH1 loop ma ki umu. Kit ma ki yubu kwede nyutu alokaloka i niang pa RC-LH1 ma rwate ki Rps. palustris, pien wa tye ka nyamo wel dano ma pat pat ma pat pat ki wa tye ki rwom ma oromo me poko peptide acel acel ki pigments ma rwate ki lipids ki quinones. Apoka poka pa jami eni nyutu ni TMH proteins adek-W ma pwod pe kinongo i RC-LH1 complex mo keken nio wa kombedi gi kelo yoo me quinone me weko quinone/quinolone loko. Kabedo mogo ma ki gwoko ma ki lwongo ni lipid ki quinone ki nongo, ki wan wa nyutu alokaloka manyen i nge keto quinone ki RC, ma twero bedo maber pi photosystem II (PSII) RC pa jami ma kwo ma tye ki oxygen. Gin ma wanongo ni miyo ngec manyen i kom kit ma quinone/quinolone mako kwede ki loko i RC-LH1 core complex pa purple phototrophic bacteria.
Me konyo kwan matut i kom jami aryo ma ki nongo i Rps. palustris, wa poko RC-LH1 acel acel ki yoo me biochemical. Gin ma ki lwongo ni protein W-deficient complex (ma ki lwongo ni ΔpufW) ki kwanyo ki i dul ma pe ki pufW gene (16), ki RC-LH1 complex acel keken aye ki twero yubu. Gin ma ki lwongo ni protein W-containing complex ni ki yubu ki dul ma ki lwongo ni strain. Gin ma ki lwongo ni protein W me dul man ki loko ki 10x His tag i C-terminus ne, wek gin ma ki lwongo ni protein W-containing complex ni ki twero ribbe maber ki pol pa protein W ma pe ki nongo ki i kom gin ma ki lwongo ni immobilizing metal. Gin ma ki poko ni ki poko ne maber (16) Affinity Chromatography (IMAC).
Kit ma ki nyutu kwede i cal 1, jami aryo ni weng tye ki dul adek ma ki lwongo ni RC (RC-L, RC-M ki RC-H) ma ki guro ki LH1 antenna. Kit ma 2.80-A tye kwede i kom gin ma pe ki protein-W nyutu 16 αβ heterodimers, ma gi yubu LH1 ma ki umu ma orumo RC, ma ki lwongo ni RC-LH116 complex. Kit ma ki yubu kwede 2.65Å pa protein-W-containing complex tye ki 14-heterodimer LH1 ma ki turo ki protein-W, ma ki lwongo ni RC-LH114-W.
(A ki B) Nyutu kom gin ma ki rubu ni. (C ki D) Rangi ma ki keto i kom yat. (E ki F) Jami ma ki neno ki i kom cytoplasmic tye ki peptides ki LH1 ma ki nyutu i cartoons, ki ki keto namba gi tung cing cawa ki i protein-W gap [ma rwate ki Rba numbering. sphaeroides complex (13)]. Pi LH1-α, kala pa dul kom ma ki lwongo ni protein ni tye yellow; pi LH1-β, kala pa dul kom ma ki lwongo ni protein ni tye bulu; pi protein-W, protein ni tye makwar; pi RC-H, en tye cyan; pi RC-L, en tye Orange; pi RC-M, magenta. Cofactors ki nyutu gi ki rods, green nyutu BChl ki BPh a molecules, purple nyutu carotenoids, ki yellow nyutu UQ10 molecules. (G ki H) neno ma ki medo i kom apoka poka pa protein-W i dul ma rom ki RC-LH114-W complex (G) ki RC-LH116 complex (H). Cofactors ki nyutu gi i kit me pongo kabedo, chelated quinone ki nyutu gi ki bulu. Kabedo ma tye i kin protein-W ki nyutu ki lain ma bulu i (G), ki bur ma tiino ma quinone/quinolol nya i kom LH116 ring ki nyutu ki lain ma col i (H).
cal 1 (A ki B) nyutu RC ma ki guro ki jami ma oyabe onyo ma ki umu pa LH1αβ heterodimers, ma acel acel mako BChl aryo ki carotenoid acel (lanyut 1, C ki D). Kwan ma okato ni onyutu ni Rps obedo dul ma ki lwongo ni LH1. I yoo me dongo pa spirulina xanthin, dul ler eni tye ki dul ma pat pat pa carotenoids (17). Ento, spiropyrroxanthin aye carotenoid ma loyo ki pek ne tye maber. Pi meno, wayero me yubu spiroxanthin i kabedo weny ma LH1 mako iye. Polypeptides ma ki lwongo ni alpha ki beta gubedo TMHs acel acel ma tye ki dul kom ma cok (lanyut 1, A, B, E, ki F). Kadi bed ni pek pa jami 17 ma odong i C-terminus pe ki neno, alpha polypeptide ni ki poko ki i Met1 me Ala46 i dul aryo ni weng. β polypeptide onongo ki dwoko piny ki Gly4 me Tyr52 i RC-LH116, ki ki Ser5 me Tyr52 i RC-LH114-W. Pe ki neno pek pa 3 onyo 4 N-terminal onyo 13 C-terminal residues (lanyut S1). Kwano rwom me pek pa jami ma ki rubu ki RC-LH1 ma ki yubu ki i kom ler ma ki lwongo ni wild-type strain onyutu ni kabedo ma orweny ni obedo adwogi me poko kin peptides magi (lanyut S1 ki S2). N-terminal formylation pa α-Met1 bene ki neno (f). Kwedo ni onyutu ni α-peptide tye ki jami ma odong fMet1 onyo Asp42/Ala46/Ala47/Ala50, ki β-peptide tye ki jami ma odong Ser2 onyo Ala53, ma tye maber ki rwom me EM ma piny.
Kit ma α-His29 ki β-His36 gi rwate kwede miyo BChls nen ki wang; αβ heterodimer acel acel rwate ki jirani ne me yubu gin ma oyabe (RC-LH114-W) onyo gin ma ki umu (RC-LH116) ma orumo RC ma ki lwongo ni exciton coupled pigment array (lanyut 1, C ki D). Ka kiporo ki 877 nm band pa RC-LH114-W, 880 nm absorption red shift pa RC-LH116 tye 3 nm (lanyut 2A). Ento, dul ma ki lwongo ni circular dichroism spectrum ni rom (lanyut 2B), ma nyutu ni kadi bed ni tye apoka poka ma nen maber i kin gin ma oyabe ki ma ki umu, kabedo pa BChls rom aroma. Redshift me absorption twero bedo adwogi me dwoko piny rwom me lyeto ki medo rwom me cung i kom closed loop (18, 19), alokaloka i pigment coupling ma oa ki i closed loop (20, 21), onyo ribbe pa adwogi aryo magi (11).
(A) Ultraviolet/visible/near-infrared absorption spectrum, ma rwom ne ki keto lanyut ki rangi ma rwate ki rwom me BPh i 775 nm. (B) Circular dichroism spectrum ma ki rwate ki BChl absorbance i 805 nm. (C ki D) Ki yero ΔA spectra ki i kare ma ki nongo ki i RC-LH114-W complex (C) ki RC-LH116 complex (D). Pi poro maber, jami weny ma ki neno ni ki dwoko gi i ∆A me −A i 0.2 ps. (E) Kit ma cytochrome c2 oxidation kwede inge keto mac i kom UQ2 ma pat pat (nen cal S8 pi ngec ma ki nongo). (F) I cell ma ki dongo i te lero ma nok, ma i kine onyo ma malo (10, 30 onyo 300μMm-2 s-1, kit meno), protein W ki RC-L ma tye i dul ma ki yubu maber ki rwom me membrane ma ki poko. Nongo rwom me protein ni ki SDS-polyacrylamide gel electrophoresis ki immunoassay (nen cal S9 pi ngec ma ki nongo). Nongo ratio ma rwate ki RC-LH114-W ma ki yubu maber. Kit ma RC-L ki protein-W me dul ni tye 1:1.
BChls ma tye i rwom me 1 i αβ14 loop ma obale pa RC-LH114-W (lanyut 1, A, C, ki E) tye cok ki RC primary donor (P) ki 6.8Å ma loyo BChls ma rom ki RC-LH116 (lanyut. 1, B, D, ki F, ki S3); kadi bed kumeno, kit ma jami aryo ni gi mako kwede jami ni nyutu ni pi RC-LH114-W ki RC-LH116, kare me loko teko me tic ki LH1 me oo i RC tye 40 ±4 ki 44±3 ps (lanyut 2). , C ki D, cal S4 ki Table S2). Pe tye apokapoka mo ma pire tek i kom cwalo jami ki i RC (lanyut S5 ki coc ma ki medo iye). Wa tamo ni rwate pa kare me cwalo teko i kin LH1 ki RC-P tye pi bor, angle ki teko ma twero bedo tye pa BChl mapol i kin LH1 loop aryo ni. Nen calo yenyo kit ma teko pa LH1 tye kwede me oo i bor ma nok loyo pe oyot loyo cwalo teko ki i kabedo ma pe opore me cito i RC. Gin ma ki lwongo ni open-loop LH1 loop i RC-LH114-W twero bedo ki lyeto ma pe pire tek i te rwom me lyeto ma piny pi nyamo kit ma ki yubu kwede, ki tye ki αβ14 ring mabor ma tye i rwom me lyeto me ot ki i rwom me rangi pa βBChls i kabedo pa RC 1.
Gin ma ki lwongo ni RC-LH116 tye ki 32 BChls ki 16 carotenoids, ki kit ma ki yubu kwede tye acel ki ma ki nongo ki i Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) DB (PDB) strain (PDB07) (297) ki lum ma lum lum (Blc.viridis) (PDB ID 6ET5) (10). Iyonge rwate, apoka poka manok keken i kabedo pa αβ heterodimers aye ki neno, tutwale 1-5, 15, ki 16 (lanyut S6). Bedo tye pa protein-W tye ki adwogi madit i kom kit ma LH1 tye kwede. TMHs adek ne gikubbe ki dul ma cok, ki N-terminal i dul ma ki lwongo ni lumen ki C-terminal i dul ma ki lwongo ni cytoplasmic (lanyut 1A ki 3, A nio wa i D). Protein-W tye ma pe maro pii (lanyut 3B), ki TMH2 ki TMH3 gi rwate ki LH1αβ-14 me yubu kom ma ki lwongo ni transmembrane surface (lanyut 3, B ki E me o i G). Gin ma ki lwongo ni interface ni tye ki Phe, Leu ki Val ma odong i dul ma ki lwongo ni transmembrane region. Jami ma odong ni ki keto gi i kom amino acid ma pe maro pii ki αβ-14 pigments. Polar residues mogo bene gi konyo i rwate, medo ki hydrogen bond i kin W-Thr68 ki β-Trp42 i kom dul ma ki lwongo ni complex cavity (lanyut 3, F ki G). I kom kom cytoplasm, Gln34 tye cok ki dul keto pa αβ-14 carotenoids. Medo ikom meno, n-dodecyl β-d-maltoside (β-DDM) molecule ni ki cobo, ki dul kome ma pe maro pii omede i kin protein-W ki αβ-14, ki dul kom ma ki lwongo ni lipid twero bedo i kom. Wa neno bene ni C-terminal resolution regions pa protein W ki RCH tye cok tutwal, ento pe tye i kin rwom me yubu rwate (lanyut 1, A ki E). Ento, twero bedo ni tye rwate i kin C-terminal amino acids ma pe ki cobo pa protein aryo magi, ma twero miyo yoo me nongo protein-W i kare me coko RC-LH114-W complex.
(A) Protein-W, ma tye ka rwate ki LH1αβ14 i kit me katun, tye ki dul ma nen calo yat (makwar), ma ki nyutu i dul me electrostatic potential diagram (kom ma nen macol ki rwom me 0.13). (B) Protein-W ma ki nyutu ki rangi ma pe maro pii. Kabedo ma tye ki polar ki ma tye ki teko ki nyutu ki rangi ma cyan, kabedo ma pe maro pii ki nyutu ki rangi ma tar, ki kabedo ma pe maro pii matek ki nyutu ki rangi ma orange. (C ki D) Protein-W ma ki nyutu i katun, kit ma ki nyutu kwede rom ki ma ki nyutu i (A) (C), ki ki loko ki 180° (D). Malube ki kama ki keto iye i dul ni, jami ma ki poko gii ni gi kwanyo rangi ma nen calo lakalatwe, ma N-terminal tye bulu ki C-terminal tye makwar. (E) Protein-W i kit ma ki neno kwede i (A), ki jami ma odong i kin protein-W:LH1 ki nyutu ki jami ma ki keto iye lanyut. (F) Protein-W ki loko 90° ma lubu (E) ki LH1αβ14 i cal ma ki nyutu i katun, ki ma lubu gin ma odong i bar. Gin ma odong ma aa ki i beta polypeptide ni ki keto nying gi iye. Gin ma ki lwongo ni cofactor ni ki nyutu calo bar ma rwate ki kala pa cal 1, β-DDM ma obale ni ki nyutu ki rangi macol, ki yamo ma ki lwongo ni oxygen ki nyutu ki rangi makwar. (G) Neno i (F) ki loko 180°, ki jami ma odong ma ki keto i kom alpha polypeptide.
Protein-W loko αβ heterodimer (me 15 i cal 1F), ma gengo loop closure ki loko αβ heterodimers adek mukwongo. Ki neno ni rwom ma malo pa αβ-1 heterodimer mukwongo ma rwate ki filim ma tye kakare onwongo tye 25° onyo 29° (lanyut 1, A ki E), ma ki yubu ki 2° onyo 8° rwom pa αβ-1 i RC A sharp contrast-LH116 (lanyut 1, B ki F). Heterodimers me aryo ki me adek gi rwate ki 12° onyo 22° ki 5° onyo 10°, kit meno. Pi tyen lok me steric hindrance pa RC, tilt pa αβ-1 pe kwako dul me aryo pa αβ (ma rwate ki 16th αβ i cal 1F), ma oweko ki yubu gap ma nen maber i LH1 ring (lanyut 1, A ki E) . Pi peke pa αβ heterodimers aryo, kacel ki rwenyo pa BChl angwen ki carotenoids aryo, pe tye carotenoids mo ma mako αβ-1 subunit ma ki loko, ma okelo LH114-W ring ma tye ki carotenoids 13 ma ki lwongo ni Vegetarian ki 28 BChls. Ki byeko rwom me rwate pa jami aryo ni i αβ1 onyo 7 tye piny loyo pa dul mukene me LH1, ma twero nyutu rwom me dongo pa LH1 ma tye cok ki RC QB site (lanyut 4).
Cal pa RC-LH114-W (A ki B) ki RC-LH116 (C ki D) ki nyutu ki i neno ma malo/ki tung cing acel (A ki B) (A ki C) ki kama ki neno iye i cal 1. (B ki D). Lagony ma ki keto kala ni ki nyutu tung acuc.
Gin acel keken ma tye ki rwom me 1:14 tye Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Ento, gin ma ki lwongo ni protein W ki PufX gi pe ki kit ma gi rwate kwede, ki gi tye ki adwogi madit i kom kit ma LH1 gi nen kwede. PufX tye TMH acel ki N-terminal cytoplasmic domain ma rwate ki cytoplasmic side pa RC-H subunit (13) i kabedo ma rwate ki Rps. palustris LH116αβ-16. PufX yubu yoo pi quinone/quinolone ma ki loko i kin RC-LH1 ki cytochrome bcl complex ki tye i Rba weny. sphaeroides core complex (13). Kadi bed ni monomer-monomer interface tye i Rba. Gin ma ki lwongo ni sphaeroides RC-LH1-PufX dimer tye i kabedo ma protein W mako iye i RC-LH114-W, ki apoka ma PufX ki protein-W okelo tye i kabedo ma rom (lanyut S7A). Kabedo ma tye i RC-LH114-W bene rwate ki quinone channel (8) pa Pseudomonas rosea LH1, ma ki yubu ki peptides ma pe kubbe ki protein W onyo PufX (lanyut S7B). Medo ikom meno, yoo me quinone ma tye i Blc. Gin ma ki lwongo ni emerald green LH1 ma ki yubu ki kwanyo γ subunit acel (7) tye i kabedo acel (lanyut S7C). Kadi bed ni ki rwate ki jami ma pat pat, nen pa jami ma ki lwongo ni quinone/quinolol i kabedo acel i RC-LH1 nen calo obedo labol me dongo ma rwate, ma nyutu ni apoka ma ki yubu ki protein W twero tic calo jami ma ki lwongo ni quinone channel.
Kabedo ma tye i LH114-W loop miyo kare me yubu dul ma rwate i kin dul ma i RC-LH114-W ki bulk membrane (lanyut 1G), ma ka kubu dul aryo ni ki i dog pore ma calo i proteins. Gin ma ki lwongo ni RC-LH116 ni rom ki Tch ma ki umu. Gin ma nen calo yat (22) (lanyut 1H). Kit ma nya pa quinone ki i membrane ni oyot loyo nya pa quinone ki i yoo ma tidi, LH114-W loop ma oyabe twero weko RC loko oyot loyo LH116 loop ma ki umu, ki nya pa quinone i RC twero bedo ma ki gengo. Me temo ka ce protein W twero gudo loko pa quinones kun wok ki i RC, watimo cytochrome oxidation assay i kom rwom me ubiquinone 2 (UQ2) (ma rom ki UQ10 ma tye ki isoprene tail ma cok) (lanyut 2E). Kadi bed ni bedo pa quinone ma ki lwongo ni chelated quinone gengo moko rwom me Michaelis constant (RC-LH114-W ki RC-LH116 gi rwate pi 0.2±0.1μM ki 0.5±0.2μM, kit meno), wel ma malo pa RC-LH114-W ( 4.6±0.6±0.2μM) tye s-1-1. 28±5% dit loyo RC-LH116 (3.6±0.1 e-RC-1 s-1).
Wa tamo ni protein-W tye i 10% pa core complex (16); kany, rwom me bedo pa ler ma nok, ler ma i kine, ki ler madwong tye 15±0.6%, 11±1% ki 0.9±0.5, kit meno % (lanyut 2F). Poro wel pa mass spectrometry onyutu ni medo histidine tag pi odwoko piny wel protein-W ka kiporo ki wild-type strains (P = 0.59), pi meno rwom magi pe obedo gin ma ki yubu ki protein-W ma ki loko (lanyut S10). Ento, rwom ma piny pa protein-W i RC-LH1 twero weko RCs mogo gi loko oyotoyot, ma weko gi dwoko piny rwom me quinone/quinolone ma tye i RC-LH116. Waneno ni rwom me lero ma malo ni pe rwate ki lok ma ki nongo i kare ni, ma nyutu ni pufW gene expression mede i te lero matek (lanyut S11) (23). Apokapoka ma tye i kin coc pa pufW ki keto protein-W i RC-LH1 ni tye ka kelo apokapoka ki twero nyutu kit ma protein ni doro kwede.
I RC-LH114-W, 6 cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) ki 29 β-DDM molecules ki poko ki ki yubu iye 6 CDLs, 24 POPCs, 2 POPGs ki 12βDs. RC-LH116 (lanyut 5, A ki B). I kin jami aryo eni, CDL tye i dul kom ma ki lwongo ni cytoplasmic, kun POPC, POPG ki β-DDM pol ne tye i dul ma ki lwongo ni luminal. Molecules aryo me lipid ki detergent ki kwanyo gi ki i αβ-1 onyo αβ-6 me RC-LH114-W complex (lanyut 5A), ki abic ki kwanyo gi i kabedo ma rom ki RC-LH116 (lanyut 5B). Ki nongo jami mapol ma ki lwongo ni lipids i tung cing mukene me dul ni, ma pol ne CDL, ma ki nongo i kin RC ki αβ-7 me oo i αβ-13 (lanyut 5, A ki B). Jami mukene ma ki yubu maber ki jami ma ki lwongo ni detergents tye woko ki i LH1 ring, ki acyl chains ma ki yubu maber gi dongo i kin LH1 subunits, ma ki ngeyo ni β-DDM i RC-LH114-W, ki ki ngeyo ni β-DDM i RC A ma ki rubu ki β-DDM ki PO-LH1616. Kabedo ma rom pa chelating lipids ki detergents i yub wa nyutu ni gin gubedo kabedo ma rwate ki kom (lanyut S12A). Kabedo pa jami ma rwate i Tch bene tye ki rwate maber. Mot ki Trv. Kit 970 RC-LH1s (lanyut S12, B onyo E) (9, 12) ki hydrogen-bonding residues me dul ma ki lwongo ni lipid head gunyutu gwok maber i rwom me rwom (lanyut S13), ma nyutu ni CDL ma ki gwoko ma mako RC (24), kabedo magi ki twero gwoko gi i RCH-LH1 complex.
(A ki B) RC-LH114-W (A) ki RC-LH116 (B) peptides ki nyutu gi ki katun, ki pigments ki nyutu ki rods, kun kitiyo ki rangi ma tye i cal 1. Lipids ki nyutu ki rangi makwar, ki detergents ki nyutu ki gray. UQ ma ki keto i RC QA ki QB tye yellow, kun UQ ma ki poko tye bulu. (C ki D) neno acel ki (A) ki (B), ki moo ma ki kwanyo woko. (E to G) Neno ma ki medo i kom Q1(E), Q2(F) ki Q3(G) ki i RC-LH116, ki dul ma gi loko i kom ngat acel acel. Gin ma ki lwongo ni hydrogen bonds ni ki nyutu calo lain macol.
I RC-LH116, gin aryo ni RC QA ki QB UQ, ma gi nywako i lok me poko electron i yub me poko teko, gi rweny woko i kabedo gi me ribbe. Ento, i RC-LH114-W, QB quinone pwod pe ki cobo ki ki bi nyamo ne matut piny kany. Medo ikom QA ki QB quinones, chelated UQ molecules aryo (ma tye i kin RC ki LH1 rings) ki poko gi i RC-LH114-W structure malube ki dul wic gi ma ki ngeyo maber (ma tye i Q1 ki Q2, kit meno). kabedo). cal 5C). Gin aryo ma ki lwongo ni isoprene ki keto i Q1, ki map me pek pa jami ni nyutu 10 isoprene tails me Q2. I kit ma RC-LH116 tye kwede, jami adek ma ki lwongo ni chelated UQ10 (Q1 onyo Q3, cal 5D) ki nongo gi, ki jami ni weng tye ki pek ma nen maber i kom yic (lanyut 5, D onyo G). I kin jami aryo ni, kabedo pa dul ma ki lwongo ni quinone head groups me Q1 ki Q2 tye ki rwate maber (lanyut S12F), ki gi rwate ki RC keken. Q1 tye i doggola me W gap pa RC-LH114-W (lanyut 1G ki 5, C, D ki E), ki Q2 tye cok ki ka ma QB mako iye (lanyut 5, C, D) ki F). Gin ma ki gwoko ma ki lwongo ni L-Trp143 ki L-Trp269 tye cok ki Q1 ki Q2 ki miyo π-stacking interactions (lanyut 5, E ki F, ki cal S12). L-Gln88, 3.0 Å ki i oxygen ma tye i Q1, miyo hydrogen bond matek (lanyut 5E); gin ma odong ni ki gwoko i RCs weny ma pat ki dul ma tye ka mabor loyo (lanyut S13). L-Ser91 ki keto i kabedo pa Thr i pol RCs mukene (lanyut S13), tye 3.8 Angstroms ki methyl oxygen me Q1, ki twero miyo hydrogen bonds ma goro (lanyut 5E). Q3 pe nen calo tye ki rwate mo, ento tye i kabedo ma pe maro pii i kin RC-M ki LH1-α subunit 5 onyo 6 (lanyut 5, D ki G). Q1, Q2 ki Q3 onyo cok ki chelated quinones bene ki cobo ne i Tch. Mot, Trv. Kit 970 ki Blc. Kit ma iris tye kwede (9, 10, 12) nyutu kabedo ma ki gwoko ma konyo quinone ma mako RC-LH1 complex (lanyut S12G). UQs abic ma obale i RC-LH116 tye i rwate maber ki 5.8±0.7 pa dul acel acel ma ki nongo ki high performance liquid chromatography (HPLC), kun UQs adek ma obale i RC-LH114-W tye piny loyo wel ma ki pimo me 6.2±0.3 (Fig. S14) nyutu ni pe tye mole ma ki nongo i UQ. kit ma tye kwede.
Gin ma ki lwongo ni pseudo-symmetric L ki M polypeptides ni tye ki TMHs abic ki gi yubu heterodimer ma gi keto BChl dimer acel, BChl monomers aryo, bacteriophage (BPh) monomers aryo, ki non- Heme iron acel ki UQ10 molecules acel onyo aryo. Ki i bedo pa hydrogen bonds i kom dul ketone ma lacen ki rwom me dongo ne i Rps, carotenoids gi donyo i M-subunit, ma ki lwongo ni cis-3,4-dehydroorhodopin. Kit ler (25). Kabedo ma woko pa RC-H ki keto i kom TMH acel. Kit ma RC ni nen kwede rom ki dul adek ma ki lwongo ni subunit RC pa ler ma gi rwate (ma calo Rba). sphaeroides (PDB ID: 3I4D). Macrocycles pa BChl ki BPh, carotenoid backbone ki non-heme iron gi rwate i kin rwom me rwom me jami eni, ma calo UQ10 head group i QA site ki QB quinone i RC-LH116 (lanyut S15).
Nongo pa jami aryo ma ki lwongo ni RC ma tye ki rwom me bedo pa QB ma pat pat miyo kare manyen me neno alokaloka ma rwate ki QB quinone binding. I RC-LH116 complex, QB quinone tye i kabedo ma ki mako maber (26), ento poko pa RC-LH114-W pe ki QB quinone. Pe tye QB quinone i RC-LH114-W, ma obedo gin me aura pien gin ma ki lwongo ni complex ni tye ka tic, makato RC-LH116 complex ki QB quinone ma ki yubu maber. Kadi bed ni LH1 rings aryo ni gi chelate ma romo quinones abicel, abic gi tye ma ki yubu maber i RC-LH116 ring ma ki umu, kun adek keken gi tye ma ki yubu maber i RC-LH114-W ring ma oyabe. Peko man ma omede ni twero nyutu ni ki loko RC-LH114-W QB oyoto, quinone kinetics oyoto i dul ni, ki medo rwom me kato ki i LH1 loop. Wa tamo ni peke pa UQ i kabedo me RC QB pa RC-LH114-W twero bedo adwogi me gin ma tek ki ma tiyo matek, ki kabedo me QB pa RC-LH114-W dong ki juku oyot i UQ turnover.
Labongo QB, loko pa L-Phe217 i kabedo ma pe rwate ki UQ10, pien obi kelo rwate ki isoprene mukwongo me yic (lanyut 6A). Medo ikom meno, alokaloka ma nen ma nen, tutwale helix de (helix matidi ma tye i kin TMH D ki E) ma L-Phe217 ki loko i QB binding pocket ki loko L-Tyr223 (lanyut 6A) me turo hydrogen bond ki M-Ap4 ki cego dul ma kubbe pa QB kabedo (lanyut 6B). Helix de pivots i tyen ne, Cα pa L-Ser209 ki loko ki 0.33Å, kun L-Val221Cα ki loko ki 3.52Å. Pe tye alokaloka mo ma nen i TMH D ki E, ma ki twero keto i kom jami aryo ni weng (lanyut 6A). Kit ma wangeyo kwede, man aye yub mukwongo i RC ma kicweyo ma umu kabedo pa QB. Ka kiporo ki dul ma opong (QB-bound) nyutu ni ma peya ki dwoko piny quinone, alokaloka me kit ma ki yubu kwede mite me weko en donyo i quinone. L-Phe217 loko me yubu π-stacking interaction ki quinone head group, ki helix loko woko, miyo skeleton pa L-Gly222 ki side chain pa L-Tyr223 me yubu hydrogen bond network ki hydrogen bond structure ma ocung matek (lanyut 6, A ki C).
(A) Cal ma ki nyutu ma rwate ki hologram (L chain, orange/M chain, magenta) ki apo (gray) structure, ma iye jami ma odong ma ki nyutu i kit ma ki nyutu kwede. UQ10 ki nyutu ki rangi ma yellow. Lain ma ki keto ni nyutu dul ma ki lwongo ni hydrogen bonds ma ki yubu i kom gin ni weny. (B ki C) Kit ma ki nyutu kwede apolipoprotein ki dul kom ma ki lwongo ni ring ni, ma nyutu dul kom ma ki lwongo ni side chain oxygen pa L-Phe217 ma tye bulu ki L-Tyr223 ma tye ki rangi makwar, kit meno. L ma ki lwongo ni subunit ni tye orange; dul M ki H pe ki rangi. (D ki E) Apolipoprotein (D) ki (E) RC QB sites [kala ki (A) kit meno] ki Thermophilus thermophilus PSII (ma lum, bulu ki plastic quinone; PDB ID: 3WU2) Align (58).
Labongo akalakala, kadi bed ni jami mapol ma tye ki QB-deficient RCs ma pe ki LH1 tye, alokaloka ma ki neno i kwan man pe ki tito con. Man medo ki kit ma ki kwanyo kwede QB ki bot Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) ki Rba. sphaeroides (PDB ID: 1OGV) (29), ma weng gi rom ki kit ma gi nen kwede. Neno 3PRC maber onyutu ni LDAO (Lauryl Dimethyl Amine Oxide) jami ma ki lwongo ni detergent molecules gi mako i doggola me QB position, ma twero gengo dwoko rwom me tic gi piny. Kadi bed ni LDAO pe balo i kabedo acel i 1EYS onyo 1OGV, RCs magi ki yubu kun kitiyo ki jami me lwoko bongo acel ki dong twero kelo adwogi acel. Kit ma ki yubu kwede Rba. Sphaeroides RC ma ki keto kacel ki cytochrome c2 (PDB ID: 1L9B) bene nen calo tye ki kabedo me QB ma ki umu. Ento, i lok man, dul N-terminal pa RC-M polypeptide (ma tye ka tic ki QB binding site kun wok ki i H bond pa Tyr residue i kom Q helix) gamo kit ma pe rwate, ki alokaloka me QB pe ki medo kwedo ne (30 ). Gin ma kweyo cwinya tye ni wan pe waneno kit alokaloka man pa M polypeptide i RC-LH114-W structure, ma cok rom ki N-terminal region pa RC-LH116 RC. Omyera bene ki nge ni ingee kwanyo gin ma ki lwongo ni detergent-based LH1 antenna, apolipoprotein RCs ma tye i PDB ni ki tyeko, ma okwanyo kin quinone ki lipids ma tye i kin RC ki dul kom ma orumo LH1 (31, 32). RC tye ka tic pien en gwoko cofactors weng, labongo decomposable QB quinone, ma pe cung matek ki pol kare rweny i kare me yubu ne (33). Medo ikom meno, ki ngeyo ni kwanyo LH1 ki jami ma ki lwongo ni natural cyclic lipids ki i RC twero bedo ki adwogi i kom tic, ma calo dwoko piny kwo pa charge-separated P+QB-state (31, 34, 35). Pi meno, wa tamo ni bedo tye pa dul LH1 ma orumo RC twero gwoko kabedo me QB ma "ki umu", ma weko gwoko kabedo macok ki QB.
Kadi bed ni apolipoprotein (labongo QB quinone) ki kit ma ki yubu kwede nyutu cal aryo keken me loko kabedo pa QB, ma ka bedo jami mapol, tye lanyut ma nyutu ni rwate ni ki twero gengo me gengo dwoko cen pa hydroquinone me gengo gengo substrate. Kit ma quinolol ki quinone gi rwate kwede cok ki QB kabedo pa apolipoprotein twero bedo pat, ma weko RC kwero ne. Ki dong ki waco ni alokaloka me kit ma jami nen kwede tye ki tic i mako ki dwoko piny jami ma ki lwongo ni quinones. Ker pa RCs ma ki juku me dwoko piny quinones ingee rwate ki piny macol tye ka rweny (36); X-ray crystallography nyutu ni balo eni oa ki i QB quinones ma ki mako i "distal" ma tye 4.5 Å ki kama tye ka tic iye (26) , 37). Wa tamo ni kit ma ki keto kwede jami ni obedo cal ma ki nyutu i kin apolipoprotein ki dul kom ma opong, ma lubo rwate ki quinone ki yabo kabedo me QB.
Gin ma ki lwongo ni type II RC ma ki nongo i PSII complex pa bacteria mogo ma gi kwo ki mac ki cyanobacteria, algae ki yadi tye ki kit ma ki gwoko kwede ki kit ma tiyo kwede (38). Kit ma ki yubu kwede ma ki nyutu i cal 6 (D ki E) nyutu rom pa PSII RCs ki kabedo me QB pa bacterial RC complex. Apoka poka ni obedo labol pi kare malac me kwan i kom jami ma mako quinone ma mako ki dwoko piny. Coc macon owaco ni alokaloka me kit ma jami nen kwede bino kacel ki dwoko piny pa PSII pa quinones (39, 40). Pi meno, ka kiporo ki kit ma RC odongo kwede, kit ma onongo peya ki neno kwede ni twero bedo ni tiyo i kom QB kabedo pa PSII RC i kom jami ma kwo ma tye ki oxygen.
Rps ΔpufW (unlabeled pufW deletion) ki PufW-His (C-terminal 10x His-tagged protein-W expressed from the natural pufW locus) strains. palustris CGA009 onongo kiloko iye i tic wa mukato angec (16). Gin ma ki lwongo ni isogenic wild-type parent ki nongo gi ki i freezer kun ki keto wel cell manok i kom PYE (ka acel acel 5 g liter -1) (ma ki gwoko i LB i -80 °C, ma tye ki 50% ( w/v) glycerol) protein, yeast extract ki succinate) agar [1.5%/w) plate. Ki keto gin ni i dyewor i piny macol i rwom me lyeto me ot ma pe ki yamo iye, ki dong ki keto mac ma ler (~50 μmolm-2 s-1) ma kimiyo ki OSRAM 116-W halogen bulbs (RS Components, UK) pi nino 3 onyo 5 nio ka colony acel onen. Ki tiyo ki dul acel me keto 10 ml me M22+ medium (41) ma ki medo ki 0.1% (w/v) casamino acids (ma dong ki lwongo ni M22). Gin ma ki pito ni ki pito i te yamo manok i piny macol i 34°C ki yenge i 180 rpm pi cawa 48, ki dong 70 ml pa gin ma ki pito ni ki keto i te piny acel-lu pi cawa 24. Gin ma ki lwongo ni semi-aerobic culture ki dwonge pa 1 ml ki tiyo kwede me keto 30 ml me M22 medium i 30 ml me universal screw-top transparent glass bottle ki ki keto i irradiation ki agitation (~50μmolm-2 s-1) pi cawa 48 ki teko me sterile magnetic force Stirring rod. Ki dong 30 ml me yat ni ki keto iye lita 1 me yat i te jami acel-lu, ma ki tiyo kwede me keto lita 9 me yat ma ki keto i ~200 μmolm-2 s-1 pi cawa 72. Ki kwanyo cell ni ki centrifugation i 7132 RCF pi dakika 30, ki dwoko i ~10 ml me 20 mM tris-HCl (pH 8.0), ki ki gwoko i -20°C nio ka mitte.
Iyonge lyel, med jami mogo ma ki lwongo ni deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) ki yat aryo ma ki lwongo ni Roche holoenzyme protease inhibitor (Merck, UK) i kom cell ma ki dwoko cen. I 20,000 psi French pressure cell (Aminco, USA), cell ni ki balo tyen 8 onyo 12. Iyonge kwanyo cell ma pe otur ki jami ma pe romo gonyo ki centrifugation i 18,500 RCF pi dakika 15 i 4°C, membrane ni ki kwanyo ki i pigmented lysate ki centrifugation i 113,000 RCF pi cawa 2 i 43,000°C. Kwany dul ma romo gonyo ni ki dwoko rangi del kom ni i 100 onyo 200 ml me 20 mM tris-HCl (pH 8.0) ki i lok nio ka jami ma nen ni pe nen. Gin ma ki keto ni ki keto i 20 mM tris-HCl (pH 8.0) (Anatrace, USA) ma tye ki 2% (w/v) β-DDM pi cawa 1 i piny macol i 4°C ki buru ne mot. Ki dong centrifuge i 70°C me juku 150,000 RCF i 4°C pi cawa 1 me kwanyo jami ma odong ma pe gonyo.
Gin ma ki lwongo ni solubilizing membrane ma aa ki i ΔpufW strain ni ki keto i kom 50 ml DEAE Sepharose ion exchange column ki column adek (CV) me binding buffer [20 mM tris-HCl (pH 8.0) ma tye ki 0.03% (w / v) β-DDM]. Lwok gin ma ki lwongo ni column ni ki CV binding buffers aryo, ki ingeye lwoko column ni ki binding buffers aryo ma tye ki 50 mM NaCl. Gin ma ki lwongo ni RC-LH116 ni ki kwanyo ki rwom me 150 onyo 300 mM NaCl (i binding buffer) i kom 1.75 CV, ki binding complex ma odong ni ki kwanyo ki binding buffer ma tye ki 300 mM NaCl i kom 0.5 CV. Kwany gin ma ki lwongo ni absorption spectrum i kin 250 ki 1000 nm, gwok dul ma tye ki absorbance ratio (A880/A280) ma kato 1 i 880 onyo 280 nm, dilute tyen aryo i binding buffer, ki tic ki yoo acel-lu i kom DEAE column On purification. Dilu dul ma tye ki A880/A280 ratios ma malo kato 1.7 ki A880/A805 ratios ma malo kato 3.0, tim dul me adek me loko ion, ki gwoko dul ma tye ki A880/A280 ratios ma malo kato 2.2 ki A880/A805 ratios ma malo kato 5.05. Gin ma ki yubu ni ki keto i ~2 ml i Amicon 100,000 molecular weight cut-off (MWCO) centrifugal filter (Merck, UK), ki ki keto i kom Superdex 200 16/600 size exclusion column (GE Healthcare, US) ma tye ki 200 mM NaCl, ki ki keto i buffer acel-lu. CV. Kwany jami ma ki nongo ki i dul ma ki kwanyo ki ikom dit, ki iket jami ma ki nongo ki ikom A880/A280 ratio ma kato 2.4 ki A880/A805 ratio ma kato 5.8 onyo 100 A880, ki cut cut tii kwede me yubu cryo-TEM grid onyo gwoko ne Gwok i -80°C nio ka mite.
Gin ma ki lwongo ni solubilizing membrane ma aa ki i PufW-His strain ni ki keto i 20 ml HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) ma tye ki 200 mM NaCl ki 0.03% (w/w)) i IMAC buffer ( GE Healthcare). v) β-DDM]. Gin ma ki lwongo ni column ni ki lwoko ki CVs abic me IMAC buffer, ki dong ki CVs abic me IMAC buffer ma tye ki 10 mM histidine. Gin ma ki lwongo ni core complex ni ki kwanyo ki i column ki IMAC buffers abic ma tye ki 100 mM histidine. Gin ma tye ki RC-LH114-W complex ni ki keto i ~10 ml i tank ma ki buru ma tye ki Amicon 100,000 MWCO filter (Merck, UK), ma ki dilute tyen 20 ki binding buffer, ki dong ki medo i 25 ml I DEAE Sepharose column, CVs angwen ma ki keto i b buffer ma ki keto i vava. Lwok gin ma ki lwongo ni column ki CV binding buffers angwen, ki dong elute complex i kom CVs aboro i kom linear gradient me 0 onyo 100 mM NaCl (i binding buffer), ki CVs angwen ma odong ma tye ki 100 mM binding buffer. Residual complexes eluted on the sodium chloride combined with the A88888/A88080 ratio and the higher than ratio. A880/A805 ratio ma malo kato 4.6 ma ki keto i ~2 ml i Amicon 100,000 MWCO centrifugal filter, ki ki pongo ki 1.5 CV IMAC ma peya Buffer equilibrated Superdex 200 16/600 size exclusion column, ki dong ki kwanyo i buffer acel-lu ma kato CV 1.5. Kwany jami ma ki nongo ki i dul ma ki kwanyo ki i kom jami ma ki kwanyo ki ikom jami ma ki kwanyo ki ikom A880/A280 ratio ma kato 2.1 ki A880/A805 ratio ma kato 4.6 onyo 100 A880, ma ki tiyo kwede cut cut me yubu grid me TEM ma ki juku onyo ki gwoko i -80°C nio ka mite.
Ki tiyo ki Leica EM GP immersion freezer me yubu TEM grids ma lyet. Gin ma ki yubu ni ki keto i IMAC buffer me A880 me 50, ki dong 5μl ki keto i kom gin manyen ma ki lwongo ni glow-discharged QUANTIFOIL 1.2/1.3 carbon-coated copper mesh (Agar Scientific, UK). Ket grid ni i 20°C ki 60% me yamo ma rwate pi 30 s, ki dong i juku ne pi 3 s, ki ingeye i juku ne i pii ma ki lwongo ni ethane i -176°C.
Lok ma ki nongo ki i RC-LH114-W complex ni ki coyo i eBIC (Electronic Bioimaging Center) (British Diamond Light Source) ki Titan Krios microscope, ma tiyo ki kero me 300kV, ki rwom me dongo pa 130,000× ki teko me Chopo-se 200V. Gatan 968 GIF Quantum ki K2 peak detector ki tiyo kwede me mako cal i kit me kwano me coko ngec. Kit ma ki pixel kwede tye 1.048Å, ki rwom me dose tye 3.83 e-Å-2s-1. Ki coko filim ni i ceken 11 ki poko ne i dul 40. Tii ki kabedo ma ki umu ki carbon me dwoko neno piny ma neno piny, ki dong i kwany filim adek i bur acel acel. Ikom weny, 3130 filim ma ki nongo, ki wel ma ki keto i kin -1 ki -3μm.
Lok ma ki nongo pi RC-LH116 complex ni ki nongo ki tic ki microscope acel-lu i Asterbury Biostructure Laboratory (University of Leeds, UK). Ki nongo ngec ni i kit me kwano ki dit pa 130 k, ki dit pa pixel ni ki rwate ki 1.065 Å ki rwom me 4.6 e-Å-2s-1. Gin ma ki nyutu ni ki mako i ceken 12 ki ki poko ne i dul 48. Ikom weny, 3359 filim ma ki nongo, ki wel ma ki keto i kin -1 ki -3μm.
Tic ki ngec weny ki tiimo i Relion 3.0 pipeline (42). Tii ki Motioncorr 2 (43) me yubu kit ma mac tye ka wot kwede ki dose weighting, ki dong tii ki CTFFIND 4.1 (44) me moko CTF (contrast transfer function) parameter. Cal ma ki kwanyo ki i kom jami ma ki lwongo ni photomicrographs ingee yub me acaki ni ki nyutu gi i cal 2. S16. Gin ma ki lwongo ni automatic selection template ni ki yubu ki yero 250 pixels pa 1000 particles i 250-pixel frame ki no reference two-dimensional (2D) classification, ki kwero poko ma rwate ki jami ma ki nyutu onyo ma pe ki kit ma nen kwede. I ngeye, ki timo yero ma otiime kene i kom cal ma ki kwanyo ni, ki RC-LH114-W onwongo tye 849,359 particles, ki RC-LH116 complex onwongo tye 476,547 particles. Jami ma ki yero ni weng gukato ki i dul aryo me non-reference 2D classification, ki inge ngwec acel acel, jami ma rwate ki kabedo me carbon, jami ma ki nyutu, ma pe ki neno onyo jami ma rwate matek ki kwero, ma oweko 772,033 (90.9%) ki 359,678 (75.5%) ma ki tiyo kwede me poko 3D 3D ma ki tiyo kwede. RC-LH114-W ki RC-LH116 kit meno. Gin ma ki lwongo ni 3D reference model ni ki yubu kun kitiyo ki yoo me stochastic gradient descent. Nono kit ma ki nyutu kwede ma calo lanyut, jami ma ki yero ni ki poko gi i dul angwen i 3D. Nono labol ma tye i dul man ma calo lanyut, tim 3D refining i kom jami ma tye i dul ma dit loyo, ki dong tic ki 15Å low-pass filter me umu kabedo ma ki lwongo ni solvent area, med 6 pixels me soft edges, ki post-process pixels me yubu Gatan K2 peak Modulation transfer function pa top detector. Pi RC-LH114-W dataset, kit ma ki cako kwede ni ki loko ki kwanyo pek pa gin ma ki lwongo ni hard density i nget mask (ma ki kwanyo ki i core complex density i UCSF Chimera). Gin ma ki nongo (kit ma RC-LH114-W ki RC-LH116 tye kwede tye 3.91 ki 4.16 Å, kit meno) ki tiyo kwede macalo lanyut pi dul me aryo me poko 3D. Jami ma ki tiyo kwede ni ki keto gi i dul me 3D ma okwongo ki pe ki rwate matek ki gang gi. Kit ma gi rwate kwede onyo pe ki jami ma nen maber. Iyonge dul me aryo me poko kin 3D, dul ma tye ki rwom ma malo loyo ki yero [Pi RC-LH114-W, dul acel tye 377,703 particles (44.5%), pi RC-LH116, tye dul aryo, ma weng tye 260,752 particles (54.7% W%, ma nongo ki keto i nge acel acel keken ka ki keto gi i nge acel acel keken i ngeye. loko ma okwongo ki apoka poka manok]. Gin ma ki yero ni ki kwanyo gi i bok ma tye ki 400-pixel ki ki yubu ki 3D. Gin ma ki lwongo ni solvent mask ni ki yubu kun kitiyo ki gin ma ki lwongo ni initial 15Å low-pass filter, 3 pixel map expansion ki 3 pixel soft mask. Nono CTF ma ki lwongo ni per-particle CTF, per-particle motion correction ki dul me aryo me per-particle CTF refinement, 3D refinement, solvent masking ki post-processing ki tiimo inge tyelo acel acel me medo rwom me rwom ma ki nongo. Nono FSC (Fourier Shell Correlation Coefficient) ma ki keto me 0.143, rwom me agiki pa RC-LH114-W ki RC-LH116 tye 2.65 ki 2.80Å, kit meno. FSC curve me agiki ma ki nyutu i cal 2. S17.
Jami ma ki lwongo ni protein ni ki kwanyo ki i 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). SWISS-MODEL (45) ki tiyo kwede me yubu homology model pa RC, ma tye ki rwom me protein pa RC-L, RC-M ki RC-H ki kit ma Rba tye kwede. sphaeroides RC onongo kitiyo kwede macalo labol (PDB ID: 5LSE) (46). Tii ki gin ma ki lwongo ni "fit map" i UCSF Chimera me keto gin ma ki yubu ni i map (47), medo rom pa protein, ki cofactor [4×BChl a (monomer library residue name = BCL), 2×BPh a (BPH), acel onyo aryo me UQ10 (U10), non-heme iron acel (Fe) ki 34 hexa-dichocaryl. (QAK)] tic ki Coot (48) me medo. Kit ma QAK pe tye i monomer library, ki keto i parameter kun kitiyo ki eLBOW tool i PHENIX (49).
Ma lubo, ki yubu dul LH1. I acaki, gin ma ki lwongo ni automatic construction tool i PHENIX (49) onwongo ki tiyo kwede me yubu dul pa LH1 kun kitiyo ki map ki LH1-α ki LH1-β protein sequences ma calo gin ma ki keto iye. Yer gin ma ki lwongo ni LH1 subunit ma otum, kwany ki i ket i Coot, med gin ma orweny iye, ki med rwom ne weny ma pwod pi i medo BCls a (BCL) ki spirilloxanthin (CRT) [ma lubu Rps ma rwate The density of the LH1 complex and the known carotenoid content. Kit ler (17)]. Coy dul LH1 ma otum, ki iti ki UCSF Chimera "Docking Map Tool" me keto i kabedo ma pe ki nyutu ma tye ki dwong pa LH1, ki dong iyubu ne i Coot; dwoko rwom me tic ni nio ka dul LH1 ni weng dong ki yubu. Pi yub me RC-LH114-W, ki kwanyo pek ma pe ki poko i Coot, protein ni ki poko ki i kom jami ma pe obedo protein ma odong i USCF Chimera map ki gin ma ki lwongo ni Autobuild ni ki tiyo kwede me yubu model mukwongo, ki dul ma odong (protein-W) Modeling. I PHENIX (49). Med gin mo kiken ma tye ka rweny i kom gin ma ki nongo i Coot (48), ki dong i lok jami ni weny. Gin ma odong ma pe ki poko ni rwate ki dul pa lipids (PDB monomer library ID me CDL = CDL, POPC = 6PL ki POPG = PGT), β-DDM detergent (LMT) ki UQ10 molecules (U10). Tii ki PHENIX optimization (49) ki manual optimization i Coot (48) me yubu model ma otum nio ka wel model ki neno maber pa gin ni pe twero medo rom ne. Me agiki ne, tii ki LocScale (50) me yubu map me kabedo moni, ki ingeye itim jami mukene mapol me yubu wel ma pe ki poko ki yubu ne kene ki cing.
Gin ma ki lwongo ni peptides, cofactors ki lipids ki quinones mukene ma ki keto i kin pek pa gi ki nyutu gi i cal 1 ki 2. S18 onyo S23. Lok ma mako wel ma ki nongo i agiki ni ki nyutu i Table S1.
Ka pi ki waco mukene, UV/Vis/NIR ma ki kwanyo ki i Cary60 spectrophotometer (Agilent, USA) i kin 1 nm ki i 250 nm nio wa i 1000 nm ki kare me keto 0.1s.
Dir gin ma ki nyutu ni i quartz cuvette ki yoo me 2 mm me A880 me 1, ki kwany rwom me nongo ne i kin 400 ki 1000 nm. Gin ma ki lwongo ni circular dichroic spectra ni ki kwanyo ki i Jasco 810 spectropolarimeter (Jasco, Japan) i kin 1 nm i kin 400 nm ki 950 nm i rwom me 20 nm min-1.
Molar extinction coefficient ki nongo ki dilute core complex me A880 ma romo 50. Dilute 10μl volume i 990μl binding buffer onyo methanol, ki kwany absorption spectrum cut cut me dwoko piny BChl degradation. Gin ma ki lwongo ni BChl ma tye i kom methanol acel acel ki kwano ki extinction coefficient i 771 nm me 54.8 mM-1 cm-1, ki extinction coefficient ki moko (51). Pok wel BChl ma ki pimo ki 32 (RC-LH114-W) onyo 36 (RC-LH116) me nongo wel core complex, ma ki tiyo kwede me nongo absorption spectrum pa gin ma ki kwanyo ki i buffer Extinction coefficient. rwate ku. Piim adek ma ki dwoko cen pi lanyut acel acel, ki rwom me nongo pa BChl Qy ma malo ni ki tiyo kwede me kwano. Gin ma ki lwongo ni extinction coefficient pa RC-LH114-W ma kipimo i 878 nm tye 3280±140 mM-1 cm-1, kun extinction coefficient pa RC-LH116 ma kipimo i 880 nm tye 3800±30 mM-1 cm-1.
UQ10 ki kwano kit ma ki tiyo kwede i (52). Ki lok macek, reverse phase HPLC (RP-HPLC) ki otimo kun kitiyo ki Agilent 1200 HPLC system. Lar maromo 0.02 nmol pa RC-LH116 onyo RC-LH114-W i 50μl pa 50:50 methanol:chloroform ma tye ki 0.02% (w/v) ferric chloride, ki i ket pre-equilibrated Beckman Coulter Ultrasphere ODS mml i 16-11-min. 40°C i HPLC solvent (80:20 methanol:2-propanol) i kom ×25 cm column. Tim isocratic elution i HPLC solvent me neno absorbance i 275 nm (UQ10), 450 nm (carotenoids) ki 780 nm (BChl) pi cawa 1. Ma malo i 275 nm chromatogram i dakika 25.5 ki keto kacel, ma onongo pe ki jami mukene ma ki twero nongo ne. Kabedo ma ki keto kacel ni ki tiyo kwede me kwano wel molar pa UQ10 ma ki kwanyo ki ikom calibration curve ma ki kwano ki i keto jami ma rwate ki 0 nio wa i 5.8 nmol (lanyut S14). Kamu kene acel acel ki kwano i tyen adek, ki bal ma ki waco ni rwate ki SD me wel ma ki nongo.
Gin ma ki lwongo ni RC-LH1 ma tye ki Qy ma malo loyo 0.1 ki yubu ki 30 μM ma ki dwoko piny horse heart cytochrome c2 (Merck, UK) ki 0 onyo 50 μMUQ2 (Merck, UK). Ki yubu kamu adek me 1-ml i rwom me UQ2 acel acel ki ki gwoko pi dyewor i piny macol i 4°C me neno ni gi rwate ki piny macol ma peya ki pimo. Lagam ni ki keto i OLIS RSM1000 modular spectrophotometer ma tye ki 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm middle ki 0.6 mm outlet slits. Gin ma ki lwongo ni 600 nm long pass filter ki keto i doggola me sample phototube ki reference photomultiplier tube me kwanyo lero ma ki lwongo ni excitation light. Kit ma ki kwanyo kwede jami ni ki neno i 550 nm ki kare me keto ne i 0.15 s. Lero ma ki lwongo ni excitation ni aa ki i 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) ki i waya ma ki lwongo ni fiber optic ma tye ki teko ma romo 90% ki i DC2200 controller (Thorlabs Ltd., UK) ki ki aa ki i kom mac ma tye ki angle me 90°. Gin ma ki pimo ni tye ka rwate ki kiyo me dwoko lero mo kiken ma onongo pe ki kwanyo ki i kom gin ma ki nyutu ni. Nen ni rwom me ngwec 10 s ma peya ki keto mac 50 s. Ki dong ki medo ki neno rwom me nongo ne pi 60 s i piny macol me neno kit ma quinolol dwoko piny kwede cytochrome c23 + (nen cal S8 pi ngec ma ki nongo).
Lok ma ki nongo ni ki tiyo kwede kun ki keto rwom me acaki ma rwate i kin 0.5 onyo 10 s (ma lubu rwom me UQ2) ki medo wel pa jami adek ni weng i rwom me UQ2 acel acel. Kit ma RC-LH1 tye kwede ma ki kwano ki rwom me rweny pa jami ni ki tiyo kwede me loko wel ne me doko rwom me tic maber, ma ki coyo i Origin Pro 2019 (OriginLab, USA), ki ki keto i model me Michaelis-Menten me moko wel Km ki Kcat ma nen.
Pi pimo rwom me nongo jami, RC-LH1 ma ki nyutu ni ki keto i ~2μM i IMAC buffer ma tye ki 50 mM sodium ascorbate (Merck, USA) ki 0.4 mM Terbutin (Merck, USA). Ascorbic acid ki tiyo kwede ma calo gin ma miyo electron, ki tert-butaclofen ki tiyo kwede macalo gin ma gengo QB me neno ni gin ma miyo RC ni odong ma nok (ma en aye, pi photooxidized) i kare me pimo ne weny. Ma romo 3 ml me kamu kene ki medo i kom gin ma ki lwongo ni custom rotating cell (ma romo 0.1 m i diameter, 350 RPM) ki 2 mm optical path length me neno ni kamu kene ma tye i yoo me laser ni tye ki kare ma oromo me loko piny macol i kin excitation pulses. Tii ki ~100-fs laser pulses me medo teko pa Ti: Sapphire laser system (Spectra Physics, USA) me juku gin ma ki nyutu ni i 880 nm i rwom me 1 kHz (20 nJ pi NIR onyo 100 nJ pi Vis). Mapwod pi i kwanyo ngec, ket lanyut ni i lero ma ki lwongo ni excitation pi dakika ma romo 30. Nyutu ne obi kelo QA ma pe tiyo (twero dwoko piny QA tyen acel onyo aryo). Ento omyera iniang ni yub man twero dwogo cen pien inge kare malac me loko kwo i piny macol, RC obi dwogo mot mot i tic pa QA. Gin ma ki lwongo ni Helios spectrometer (Ultrafast Systems, USA) ki tiyo kwede me pimo jami ma ki lwongo ni transient spectra ki kare me -10 onyo 7000 ps. Tii ki jami ma ki lwongo ni Surface Xplorer (Ultrafast Systems, USA) me poko kin jami ma ki coyo ni, ki dong i rub ki i rwom acel. Tii ki CarpetView software package (Light Conversion Ltd., Lithuania) me tic ki jami ma ki keto kacel me nongo spectra ma pat pat ma kubbe ki decay, onyo tii ki tic ma kwako exponents mapol ki lagam pa gin ni me rwate ki single-wavelength spectral evolution i Origin (OriginLab, USA).
Kit ma kiloko kwede malo ni (53), ki yubu gin ma ki lwongo ni photosynthetic film ma tye ki LH1 ma pe ki RC ki peripheral LH2 antenna. Gin ma ki lwongo ni membrane ni ki dilute i 20 mM tris (pH 8.0) ki dong ki keto i quartz cuvette ki 2 mm optical path. Ki tiyo ki 30nJ laser pulse me juku gin ma ki nyutu ni i 540 nm ki kare me -10 onyo 7000 ps. Tii ki ngec ma ki keto ni kit ma ki waco kwede pi Rps. labol pa lurem.
Gin ma ki lwongo ni membrane ni ki juku ki centrifugation i 150,000 RCF pi cawa 2 i 4°C, ki dong absorbance ne i 880 nm ki dwoko i 20 mM tris-HCl (pH 8.0) ki 200 mM NaCl. Nyaa gin ma ki lwongo ni membrane ni ki buru ne mot mot i 2% (w/v) β-DDM pi cawa 1 i piny macol i 4°C. Gin ma ki nyutu ni ki keto i 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) me oo i rwom me protein ma tye 2.5 mg ml-1 (Bio-Rad analysis). Medo tic ki yoo ma ki coyo con ni (54), cake ki keto 50 μg me protein i 50 μl TEAB ma tye ki 1% (w/v) sodium laurate (Merck, UK). Iyonge 60 s, ki dwoko piny ki 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) i 37°C pi dakika 30. Pi S-alkylation, juku gin ma ki nyutu ni ki 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) ki med ki i 200 mM isopropanol stock solution pi dakika 10 i rwom me lyeto me ot. Ki medo 2 μg trypsin/endoproteinase Lys-C mixture (Promega UK) ki ki keto i 37°C pi cawa 3. Laurate surfactant ni ki kwanyo ki medo 50 μl ethyl acetate ki 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) ki vortexing pi 60 s. poko kin jami ni ki medo ki centrifugation i 15,700 RCF pi dakika 5. Malube ki cik pa luyub, ki tiyo ki C18 spin column (Thermo Fisher Scientific, UK) me kwanyo ki kwanyo kadu i dul ma piny ma tye ki peptide ni. Iyonge juku ki vacuum centrifugation, gin ma ki nyutu ni ki juku ne i 0.5% TFA ki 3% acetonitrile, ki 500 ng ki kwano ki nanoflow RP chromatography kacel ki mass spectrometry kun kitiyo ki jami ma ki waco con ni.
Tii ki MaxQuant v.1.5.3.30 (56) me nongo protein ki kwano wel me yenyo Rps. palustris proteome database (www.uniprot.org/proteomes/UP000001426). Gin ma ki lwongo ni mass spectrometry proteomics ni ki keto i ProteomeXchange Alliance kun wok ki i PRIDE partner repository (http://proteomecentral.proteomexchange.org) ite dataset identifier PXD020402.
Pi nyamo ki RPLC kacel ki electrospray ionization mass spectrometry, RC-LH1 complex ni ki yubu ki i wild-type Rps. Nono yoo ma ki coyo con ni (16), wel protein ma ki yubu i palustris cells onwongo tye 2 mg ml-1 i 20 mM Hepes (pH 7.8), 100 mM NaCl ki 0.03% (w/v) β- (Bio-Rad analysis) ) DDM. Malube ki cik pa luyub, tii ki jami ma ki lwongo ni 2D purification kit (GE Healthcare, USA) me kwanyo 10 μg me protein ki yoo me precipitation, ki juku precipitate ni i 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile ki 20% (v/v) pii. Microliters abic ki kwano ki RPLC (Dionex RSLC) kacel ki mass spectrometry (Maxis UHR-TOF, Bruker). Tii ki MabPac 1.2×100 mm column (Thermo Fisher Scientific, UK) me poko i 60°C ki 100μlmin -1, ki rwom me 85% (v / v) solvent A [0.1% (v / v) FA ki 0.02% (V/v) TFA aqueous solution] to solvent (v / v) 85% Bv/v) [0.1%(v/v) FA ki 0.02%(v/v) i 90%(v/v) acetonitrile TFA] Nono rwom me electrospray ionization source ki default parameters pi kare ma kato dakika 60, mass spectrometer nongo 100 onyo 2750 m/z (mass-to-charge ratio). Ki kony pa ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), ket mass spectrum i dul matino tino me complex.
Gin ma ki lwongo ni cell ni ki dongo pi cawa 72 i te 100 ml NF-low (10μMm-2 s-1), medium (30μMm-2 s-1) onyo high (300μMm-2 s-1) mac. M22 medium (M22 medium ma ki kwanyo iye ammonium sulfate ki sodium succinate ki loko ki sodium acetate) i 100 ml screw-top bottle (23). I kare me 30-s abic, 0.1 micron ma ki lwongo ni glass beads ki keto i volume ratio me 1:1 me poko cell ki ki keto i kom ice pi dakika 5. Gin ma pe romo gonyo, cell ma pe otur ki jami ma ki lwongo ni glass beads ki kwanyo gi ki centrifugation i 16,000 RCF pi dakika 10 i benchtop microcentrifuge. Gin ma ki lwongo ni membrane ni ki poko i Ti 70.1 rotor ki 100,000 RCF i 20 mM tris-HCl (pH 8.0) ki 40/15% (w/w) sucrose gradient pi cawa 10.
Kit ma kiloko kwede i tic wa mukato ni, nongo two ma ki lwongo ni His tag i kom PufW (16). Ki lok macek, gin ma ki lwongo ni purified core complex (11.8 nM) onyo membrane ma tye ki dwong pa RC (ma ki nongo ki oxidation ma kwanyo apoka poka ma ki dwoko piny ki rwate ki pek pa gin ma ki keto i kom gel) i 2x SDS loading buffer (Merck, UK) ma ki juku tyen aryo. Gin ma ki lwongo ni proteins ni ki poko gii i kom gin ma ki lwongo ni replica 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). Ki keto rangi i kom gel ki Coomassie Brilliant Blue (Bio-Rad, UK) me keto ki neno RC-L subunit. Gin ma ki lwongo ni protein ma tye i gel me aryo ni ki keto i methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) pi immunoassay. Gin ma ki lwongo ni PVDF membrane ni ki gengo i 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 ki 5% (w / v) ma ki kwanyo ki i cak ma ki lwongo ni skimmed milk powder, ki dong ki gwoko ki anti-His primary antibody (i Dilute the antibody buffer [pH 7.6 7.6), 150 mM NaCl ki 0.05% (v/v) Tween-20] i 1:1000 A190-114A, Bethyl Laboratories, USA) pi cawa 4. Iyonge lwoko tyen 3 pi dakika 5 i antibody buffer, membrane ni ki rubu ki horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (ma ki keto 1:10,000 i antibody buffer) Incubate me weko nongo (dakika 5 ingee lwoko 3 i antibody buffer) kun kitiyo ki WAR EST 2000. chemiluminescence substrate (Cyanagen, Italy) ki Amersham Imager 600 (GE Healthcare, UK).
Ki goyo rwom me poko pa gel ma ki keto rangi iye onyo yoo me immunoassay, medo kabedo ma tye i te peak ki kwano rwom me teko pa RC-L (stained gel) ki Protein-W (immunoassay), i ImageJ (57) Yub cal ni. Ratio magi ki loko gi me doko molar ratio kun ki tamo ni ratio pa RC-L ki protein-W i RC-LH114-W ma ki kwanyo ni obedo 1:1 ki dwoko rwom me ngec ni weny.
Pi jami ma ki medo i kom pwony man, nen http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
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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, Andrew C. Necoil Hunter.
Kit ma ki yubu kwede light trap 1 ma tye i reaction center miyo ngec manyen i kom kit ma quinone tiyo kwede.
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, Andrew C. Necoil Hunter.
Kit ma ki yubu kwede light trap 1 ma tye i reaction center miyo ngec manyen i kom kit ma quinone tiyo kwede.
©2021 Dul pa American Association for the Advancement of Science. twero weng ki gwoko. AAAS tye larem pa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef ki COUNTER. ScienceAdvances ISSN 2375-2548.