Kawoni†Kabedo ma kawoni: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Reaction center light-harvesting complex 1 (RC-LH1) utiye piny ma pire tek ma timo photosynthesis pa purple phototrophic bacteria. Waketho cryo-electron microscopy structures ario pa RC-LH1 complex kud i Rhodopseudomonas palustris. Kit 2.65-Å resolution structure pa RC-LH114-W complex utiye ku 14 subunit LH1 loops ma ucungo ikum RC, ma jupoko ku protein W, man complex ma epe ku protein-W utiye RC composition ma ucungo ikum RC. Cwalo 16 subunit LH1 loop. Poro pa kit piny eni mio nyang ikum kit ma quinone timo ku tic i RC-LH1 complex, ikinde utiye aloka loka ma con ungiyure ngo ikare ma quinone ucungo i RC QB site, man kabedo pa auxiliary quinone binding sites, ma konyo ni tero gi i RC. Kite ma W protein utiye kudu ukwero cego pa LH1 loop, man eketho yore mi medo rwom pa quinone/quinolone.
Ker ma wok kud i photosynthesis copo gwoko kwo zo iwi ngom, man etiye ku copo madit pi biotechnology pa ceng. I kare ma jubecwaku yub pa ler mi ng'om zoo, bacteria ma rangi pa ler macol de nyutho kit tego matung tung man kero migi mi loko piny. Gicopo jwigo photosynthesis man gidongo calu heterotrophic bacteria i mudho, gicopo yiko nitrogen man carbon dioxide, giwodho hydrogen, man ginyayu aromatic compounds (1-3). Pi miyo kero ni tic eni, ler umitu julok pio pio man maber ni kero pa kemikol. Tic eni caku kinde ma antenna ma mako ler utingo ler man ecwalo kero ma mako i reaction center (RC), man eca caku poko pa mac (4 – 7). Piny ma pire tek mi photosynthesis i purple phototrophic bacteria utiye type 2 RC, ma ucungo iwi light-harvesting complex 1 (LH1), ma uketho RC-LH1 core complex. LH1 juyike ku dul pa αβ heterodimers, acel acel ma mako chlorophyll (BChl) a molecules man carotenoids acel nyo aryo (8-12). LH1 antenna ma yot uloyu utiye ku 16 nyo 17 αβ heterodimers ma ucungu RC (9-13) i loop ma cego, ento i core complexes mukende, transmembrane peptides turo rwom pa LH1, ma uketho quinol/quinone diffusion ikind RC ku cytochrome bc complex (1111, 13). Yath ma rangi pa polo ma julwongo ni Rhodopseudomonas (Rps.) ubedo piny ma kwo maber ma copo nyang ikum tego man rwom pa electron ma konyo tic pa ler. Kit ma kidi mir acel pa Rps. Lanyuth pa palustris RC-LH1 ubedo RC, ma juguro ku 15 heterodimeric LH1 loops, ma jupoko ku protein ma jungeye ngo ma julwongo ni "Protein W" (14). Protein-W ungiyure i ngeye ni RPA4402, ma en e 10.5kDa protein ma jukwanu ngo ku transmembrane helices adek (TMH) (16). Wabemito walok nying rpa4402 gene ma ketho protein W ni pufW kara ebed marom ku nying ma jutiyo kudu pi gene ma ketho RC-L, M (pufL, pufM) man LH1α, β (pufA, pufB) subunits. Ber pa lembe, protein-W utiye kende i 10% pa RC-LH1, ma nyutho Rps. palustris nyayu RC-LH1 ario matung tung. Kae, wabe nyutho rwom ma malu pa cryo-EM (cryo-EM) pa dul aryo, acel ku protein W man 14 αβ heterodimers, acel ku protein W man 16 Heterodimer LH1 loop ma cego. Kit yub mwa nyuthu aloka loka madit mi nyang ikum RC-LH1 complex pa Rps. palustris, kum wa ngiyo wel pa dhanu ma tung tung man wabedo ku rwom ma romo mi poko peptide acel acel man pigments ma rwate man lipids man quinones. Poro pa kit piny eni nyutho nia TMH adek-W ma junwange ngo i RC-LH1 complex moko cii kawoni gi kelo quinone channel mi medo quinone/quinolone exchange. Kabedo mapol ma jugwoko ma lipid ku quinone gicokiri i iye jular junwango, man wanwango aloka loka manyen i ngei rwom pa quinone ku RC, ma copo bedu ber pi photosystem II (PSII) RC pa piny ma kwo ma nwango oxygen. Lembe ma wanwango umiyu ngec manyen ikum kit ma quinone/quinolone mako man lokore i RC-LH1 core complex pa purple phototrophic bacteria.
Pi konyo somo matuc pa dul ario ma nwangre i Rps. palustris, wapoko RC-LH1 acel acel ku yore mi biochemical. Protein W-deficient complex (ma julwongo ni ΔpufW) juyike kud i strain ma epe ku pufW gene (16), man RC-LH1 complex acel kende re ma jucopo yubu. Protein W-containing complex juyubu ku kit moko. Protein W pa kit eni juyike ku 10x His tag i C-terminus pare, kara protein W-containing complex ecopu bedu maber ku protein W ma epe ku immobilizing metal. The complex is effectively separated (16) Affinity Chromatography (IMAC).
Calu ma ju nyuthu i cal mir acel, dul ario zoo gitiye ku RC adek (RC-L, RC-M man RC-H) ma juguro ku LH1 antenna. Kit 2.80-A pa dul ma epe ku protein-W nyutho 16 αβ heterodimers, ma ketho LH1 loop ma ucungo ikum RC, ma kawoni julwongo ni RC-LH116 complex. Kit 2.65Å pa protein-W-containing complex utiye ku 14-heterodimer LH1 ma jupoko ku protein-W, ma kawoni julwongo ni RC-LH114-W.
(A ku B) Nyuthu pa piny ma jucwalo. (C ku D) Rangi ma jukubothre ma ju nyuthu i rods. (E ku F) Piny ma nen kud i wi cytoplasm utiye ku peptides man LH1 subunits ma nen i cartoons, man jukwanu gi clockwise kud i protein-W gap [ma rwate ku Rba numbering. sphaeroides complex (13)]. Pi LH1-α, rangi pa dul pa protein ubedo yellow; pi LH1-β, rangi pa dul pa protein ubedo bulu; pi protein-W, protein ubedo makwar; pi RC-H, etiye cyan; pi RC-L, etiye Orange; pi RC-M, magenta. Cofactors nyuthu ku rods, green nyuthu BChl man BPh a molecules, purple nyuthu carotenoids, man yellow nyuthu UQ10 molecules. (G ku H) Neno madit pa apoka poka pa protein-W in kabedo marom pa RC-LH114-W complex (G) man RC-LH116 complex (H). Cofactors nenre i ayi mi pongu kabedo, chelated quinone nenre i rangi macol. The protein-W gap unyuthire ku blue dashed line in (G), man holes ma nok kama quinone/quinolol diffuses iwi LH116 ring unyuthire ku black dashed line in (H).
Cal 1 (A ku B) nyutho RC ma jugoro ku dul ma oyabere nyo ma cego pa LH1αβ heterodimers, acel acel ma mako BChl aryo man carotenoid acel (cal 1, C ku D). Somo mukadho unyutho nia Rps utiye LH1 complex. I yore mi yubu pa spirulina xanthin, lei eni gitiye ku dul pa carotenoids (17). Ento, spiropyrroxanthin ubedo carotenoid madit man pek pare ber. Pieno, wayero ni model spiroxanthin i kabedo ceke ma LH1 ucungo i iye. Alpha ku beta polypeptides gibedo TMHs acel ku membrane ma nok (lanyut 1, A, B, E, man F). Kadi bende rwom pa 17 residues i C-terminus unen ungo, alpha polypeptide jupoko kud i Met1 ni Ala46 i complexes ario zoo. β polypeptide judwoko piny kud i Gly4 ni Tyr52 in RC-LH116, man kud i Ser5 ni Tyr52 in RC-LH114-W. Pek pa 3 nyo 4 N-terminal nyo 13 C-terminal residues juneno ngo (lanyut S1). Mass spectrometry analysis pa mixed RC-LH1 complex ma juyubu kud i wild-type strain unyutho nia kabedo ma rwinyo ubedo adwogi pa heterologous cleavage pa peptides eni (Figure S1 ku S2). N-terminal formylation pa α-Met1 de juneno (f). Lembe ma jutimo unyutho nia α-peptide utiye ku residues fMet1 nitundo Asp42/Ala46/Ala47/Ala50, man β-peptide utiye ku residues Ser2 nitundo Ala53, ma rwate maber ku low-temperature EM density map.
Dikiri pa α-His29 ku β-His36 weko BChls bedo wang ku wang; αβ heterodimer acel acel cokiri ku judhugola pare mi yubu loop ma oyabere (RC-LH114-W) nyo loop ma cego (RC-LH116) ikum RC The exciton coupled pigment array (Figure 1, C and D). Ka juporo ku 877 nm band pa RC-LH114-W, 880 nm absorption red shift pa RC-LH116 utiye 3 nm (lanyut 2A). Ento, circular dichroism spectrum utiye marom (lanyut 2B), ma nyutho nya kadi nya tung tung utiye ikind open ku closed loops, kabedo pa BChls utiye marom. Absorption redshift copo bedu adwogi pa dwoko piny pa thermal motion man medo bedo maber pa closed loop (18, 19), aloka pa pigment coupling ma closed loop ukelo (20, 21), nyo dul pa adwogi aryo eni (11).
(A) Ultraviolet/visible/near-infrared absorption spectrum, the peaks of which marked with the corresponding pigments and normalized to the BPh peak at 775 nm. (B) Circular dichroism spectrum normalized to BChl absorbance at 805 nm. (C ku D) ΔA spectra ma juyeru kud i spectra mi RC-LH114-W complex (C) man RC-LH116 complex (D). Pi poro maber, spectra zo judwoko ni ∆A pa −A i 0.2 ps. (E) Kadhiri pa cytochrome c2 oxidation ingey irradiation i bedo pa rwom matung tung pa UQ2 (nen cal S8 pi lembe ma nyen). (F) I cell ma dongo i the lero ma nok, ma rwom pare tiye nyo ma malo (10, 30 nyo 300μMm-2 s-1, kubang wadi), protein W man RC-L subunits i purified complex man separated membrane ratio. Nwangu rwom pa protein ku SDS-polyacrylamide gel electrophoresis man immunoassay (nen cal S9 pi lembe ma nyen). Nwangu ratio ma rwate ku RC-LH114-W complex ma juyubu. Ratio pa RC-L ku protein-W pa complex utiye 1:1.
BChls i kabedo mir acel i αβ14 loop pa RC-LH114-W (lanyut 1, A, C, man E) gi ceng ku RC primary donor (P) ku 6.8Å ma sagu BChls ma rom i RC-LH116 (lanyut 1, B, D, man F, man S3); ento, rwom mi mako piny ma nyai pa dul aryo eni nyutho nia pi RC-LH114-W man RC-LH116, rwom pa kero mi nyayu piny kud i LH1 ni RC utiye 40 ±4 man 44±3 ps (lanyut 2). , C ku D, cal S4 man Table S2). Bende epe tung tung madit ikum cwalo piny i RC (lanyut S5 man lembe mange ma rwate). Waparu niya rwate pa kare mi poko tego ikind LH1 ku RC-P utiye pi bor marom, angle man tego pa BChl mapol i LH1 loops aryo. Nen calu ni sayu kit pa kero pa LH1 ni tundo bor ma nok epe nya yot uloyu rwom pa kero ma wok kud i kabedo ma ber ongo ni RC. Open-loop LH1 loop in RC-LH114-W copo bende kadhu kud i thermal motion ma nok i thenge mi lyethu ma piny pi nyang ikum kite, man etiye ku αβ14 ring conformation mabor i temperature mi ot kud i bor pa rangi pa βBChls i kabedo pa RC 1.
RC-LH116 complex utiye ku 32 BChls man 16 carotenoids, man kit ma juyike kudu utiye marom ku ma ju nwango kud i Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) strain (PDB) PDB077) man green algae (Blc.viridis) (PDB ID 6ET5) (10). I ngei ketho piny, apoka poka manok kende i kabedo pa αβ heterodimers re ma juneno, asaga 1-5, 15, man 16 (lanyut S6). Bedo tiye pa protein-W utiye ku adwogi madit ikum kit ma LH1 utiye kudu. TMHs adek pare gicokiri ku dul ma nok, ku N-terminal i thenge pa lumen pa complex man C-terminal i thenge pa cytoplasmic ( cal 1A ku 3, A ku D). Protein-W utiye piny ma maru pii (lanyut 3B), man TMH2 ku TMH3 timo tic ku LH1αβ-14 mi yubu transmembrane surface (lanyut 3, B ku E nitundo G). Ikind piny eni utiye ku Phe, Leu man Val residues in transmembrane region. Jam piny eni juporo ku amino acids ma maru pii man αβ-14 pigments. Polar residues moko de konyo i timo tic, ikinde hydrogen bond ikind W-Thr68 ku β-Trp42 iwi ngete pa complex cavity (lanyut 3, F ku G). I wi cytoplasm, Gln34 utiye ceng ku keto group pa αβ-14 carotenoids. Mi medo, n-dodecyl β-d-maltoside (β-DDM) molecule juyike, man yibe pare ma maru pii umedire nitundo i kind protein-W ku αβ-14, man yibe pa lipid copo bedu i kum. Waneno bende niya C-terminal resolution regions pa protein W ku RCH utiye kago, ento epe i thenge mi yubu interactions moko (lanyut 1, A ku E). Ento, copu bedu ku winjiri ikind C-terminal amino acids pa proteins aryo eni, ma copo mio yore mi lwongu protein-W i saa mi coko pa RC-LH114-W complex.
(A) Protein-W, ma nen ku LH1αβ14 i ayi pa cartoon, utiye ku dul ma nen calu rod (makwar), ma nen i dul pa electrostatic potential diagram (ngom macol ma nen maleng ku rwom pa 0.13). (B) Protein-W ma nyutho rangi ma rwinyo pii. Kabedo ma polar man ma charged nen i cyan, kabedo ma hydrophobic nen i white, man kabedo ma hydrophobic tek nen i orange. (C ku D) Protein-W ma ju nyuthu i katun, nen pare utiye marom ku (A) (C), man juloko ku 180° (D). Nimakere ku kabedu migi i rwom, piny ma juporo kudu nen calu rangi pa polo, kama N-terminal ubedo bulu man C-terminal ubedo makwar. (E) Protein-W in nen marom ku (A), man residues i interface pa protein-W:LH1 ju nyuthu ku rods ku lanyuth. (F) Protein-W juloko 90° malube ku (E) man LH1αβ14 i lanyuth pa katun, man malube ku piny madong i lanyuth pa bar. Lembe ma udong ma ucungo iwi beta polypeptide jukiewo nying gi. Cofactor nen calu bar ma rom ku rangi pa cal mir acel, β-DDM ma utur nen i rangi macol, man oxygen nen i rangi makwar. (G) Nen in (F) juloko 180°, ku dul ma nen pa alpha polypeptide.
Protein-W loko αβ heterodimer (ma mir 15 in cal mir 1F), ma uketho loop closure man eketho αβ heterodimers adek ma kwong. Junwango ni rwom ma malu pa αβ-1 heterodimer mir acel ma rwate ku film normal ubedo 25° ku 29° (lanyut 1, A ku E), ma juyubu ku 2° ku 8° pa αβ-1 in RC A sharp contrast-LH116 (lanyut 1, B ku F). Heterodimers mir aryo ku mir adek ucungo i 12° ku 22° man 5° ku 10°, kubang wadi. Ni lembe pa steric hindrance pa RC, tilt pa αβ-1 uketho ngo pair mir aryo pa αβ (ma rwate ku 16th αβ i cal 1F), eno uketho gap maleng i LH1 ring (cal 1, A ku E) . Ni kadhu pa αβ heterodimers aryo, ku rweny pa BChl angwen man carotenoids aryo, carotenoids moko ope ma mako αβ-1 subunit, ma ukelo LH114-W ring ma tingo carotenoids 13 Vegetarian man BChls 28. Lembe ma jukoro ikum dul ario ma nwangire i αβ1 ku 7 utiye piny uloyu ikum LH1 loop, ma copo nyutho kit ma LH1 utiye kudu ikum kabedo pa RC QB (lanyut 4).
Cal pa RC-LH114-W (A ku B) man RC-LH116 (C ku D) ju nyuthu gi kud i nen ma malu/nen ma yo piny (A ku B) (A ku C) man nen pa wang cal mir acel. (B ku D). Jam rangi ma tung tung nen yo cing acam.
Piny acel kende ma pire tek ku rwom pa 1:14 utiye Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Ento, protein W ku PufX gipe ku homology ma nen kamaleng, man gitiye ku adwogi madit ikum kit LH1 migi. PufX ubedo TMH acel ku N-terminal cytoplasmic domain ma timo tic ku cytoplasmic side pa RC-H subunit (13) i kabedo ma rwate ku Rps. palustris LH116αβ-16. PufX cwio yore mi loko quinone/quinolone ikind RC-LH1 ku cytochrome bcl complex man etiye i Rba zo. sphaeroides core complex (13). Kadi bende dul pa monomer-monomer utiye i Rba. Sphaeroides RC-LH1-PufX dimer utiye i kabedu pa protein W in RC-LH114-W, man apoka ma PufX ku protein-W ukelo utiye i kabedu marom (Figure S7A). Kabedo ma RC-LH114-W urombo ku quinone channel (8) pa Pseudomonas rosea LH1, ma juyike ku peptides ma rwate ngo ku protein W nyo PufX (Figure S7B). Mi medo, quinone channel in Blc. Emerald green LH1 ma juyubu ku weko γ subunit acel (7) utiye i kabedu marom (lanyut S7C). Kadi bende jutimo ku protein matung tung, nen pa quinone/quinolol channels eni i kabedo acel i RC-LH1 nen calu lanyuth pa convergent evolution, ma nyutho nia gap ma juyiko ku protein W copo timo tic calu quinone channel.
Gap in LH114-W loop weko bedo pa membrane region ma mediri ikind kabedo ma iye pa RC-LH114-W complex ku bulk membrane (Figure 1G), kakare ni mako domains aryo eno nikadhu kud i protein pore calu i proteins. RC-LH116 utiye marom ku Tch ma cego. Piny ma calu sindhani (22) (lanyut 1H). Calu nyai pa quinone nikadhu kud i membrane utiye nyap uloyu nyai kud i yo ma nyap pa protein, LH114-W loop ma oyabere copo ketho RC lokore pio pio uloyu LH116 loop ma cego, man nyai pa quinone i RC copo bedu tek. Pi ni neno ka nyo protein W copo loko quinones nikadhu kud i RC, watimo cytochrome oxidation assay iwi rwom moko pa ubiquinone 2 (UQ2) (analogue pa UQ10 ma ku isoprene tail ma nok) (Figure 2E). Kadi bende bedo pa chelated quinone nyayu peko i kum nyang pa Michaelis constant (RC-LH114-W man RC-LH116 gipore pi 0.2±0.1μM man 0.5±0.2μM, kubang wadi), wel ma malu pa RC-LH114-W ( 4.6±0.6±0.2-2) utiye s-1-1-. 28±5% malac uloyu RC-LH116 (3.6±0.1 e-RC-1 s-1).
I acaki, waparu niya protein-W utiye i 10% pa core complex (16); keni, wel mi bedo pa ler ma nok, ler ma ikin piny, man ler ma lee utiye 15±0.6%, 11±1% man 0.9±0.5, kubang acel acel % (lanyut 2F). Poro pa mass spectrometry unyutho nia medu pa histidine tag udwoko piny ngo wel pa protein-W ka juporo ku wild-type strains (P = 0.59), ento wel eni epe ku piny ma juloko pa protein-W (Figure S10). Ento, bedo piny ma nok pa protein-W i RC-LH1 copo ketho RCs moko lokore pio pio, ma copo dwoko piny quinone/quinolone ma nok i RC-LH116. Waneno nia rwom ma malu pa lero urombo ngo ku lembe ma nyen ma jukoro i transcriptomics, ma nyutho nia pufW gene expression medre i the lero matek (Figure S11) (23). Tung tung ma nuti kind pufW transcription ku protein-W ma juketho i RC-LH1 complex utiye lembe ma nyang ikume man ecopu nyutho kit ma protein ne ucungo kudu.
I RC-LH114-W, 6 cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) man 29 β-DDM molecules jupoko man juyubu i iye 6 CDLs, 24 POPCs, 2 POPGs man 12βDs. RC-LH116 (lanyut 5, A ku B). I kind kit ario eni, CDL utiye i thenge mi cytoplasmic pa complex, man POPC, POPG man β-DDM utiye i thenge mi luminal. Molecule ario mi lipid man detergent jupoko gi i αβ-1 ku αβ-6 mi RC-LH114-W complex (lanyut 5A), man abic jupoko gi i theng RC-LH116 (lanyut 5B). Lipids madwong junwange i thenge mange mi complex, mandha ne CDL, ma jucoko ikind RC ku αβ-7 ni αβ-13 (lanyut 5, A ku B). Piny mukende ma juyiko maber man jam lwoko piny utiye woko pa LH1 ring, man acyl chains ma juyiko maber ucungo ikind LH1 subunits, ma julwongo ni β-DDM in RC-LH114-W, man juloko ni β-DDM in RC A mixture of β-DDM and PO-LH166. Kabedo marom pa chelating lipids man detergents i yik mwa nyutho nia gitiye kabedo ma rwate ku kum (lanyut S12A). Kabedo pa molecule marom i Tch de utiye ku rwom maber. Mol man Trv. Strain 970 RC-LH1s (Figure S12, B to E) (9, 12) man the hydrogen-bonding residues of lipid head group onyuthu bedo maber i rwom mi rwom (Figure S13), ma nyutho nia CDL ma jugwoko ma mako RC (24), kabedo eni copo bedu i RCH-LH1 complex.
(A ku B) RC-LH114-W (A) man RC-LH116 (B) peptides ju nyuthu gi ku cartoons, man pigments ju nyuthu ku rods, nitio ku rangi ma nen i cal mir acel. Lipids ju nyuthu ku rangi makwar, man detergents ju nyuthu ku rangi macol. UQ ma mako RC QA man QB ubedo yellow, man UQ ma ucungo kende ubedo blue. (C ku D) Nen marom ku (A) ku (B), ku lipids ma juweko. (E to G) Neno madit pa Q1(E), Q2(F) man Q3(G) kud i RC-LH116, ku cing cing ma nyayu aloka loka ikindgi. Hydrogen bonds nen calu rangi macol.
I RC-LH116, RC QA ku QB UQ, ma gikonyo i poko electron i yub mi poko piny, gipodho i kabedu migi mi mako. Ento, in RC-LH114-W, QB quinone ungiyure ngo man jubi weco iwie piny eno. Mi medo ikum QA ku QB quinones, chelated UQ molecules aryo (ma nwangire ikind RC ku LH1 rings) jupoko gi i RC-LH114-W structure nimakere ku dul wigi ma juyiko maber (ma nwangire i Q1 ku Q2, kubang wadi). kor polo). Cal 5C). Isoprene ario jupoko ni Q1, man density map loko 10 isoprene tails pa Q2. I kit pa RC-LH116, UQ10 molecules adek ma chelated (Q1 ku Q3, cal 5D) juyubu, man molecules ceke gitiye ku pek ma nen kamaleng i ngeye (lanyut 5, D ku G). I kind kit ario eno, kabedo pa quinone head groups pa Q1 ku Q2 utiye ku rwom maber (lanyut S12F), man gi timo tic ku RC kende. Q1 utiye i dhugola pa W gap pa RC-LH114-W (lanyut 1G ku 5, C, D man E), man Q2 utiye inget kama QB ucungo i iye (lanyut 5, C, D) man F). L-Trp143 man L-Trp269 ma jugwoko gibedo kago ku Q1 ku Q2 man gimio π-stacking interactions (lanyut 5, E ku F, man cal S12). L-Gln88, 3.0 Å kud i distal oxygen pa Q1, mio ​​hydrogen bond matek (lanyut 5E); residue eni jugwoko i RCs zo mako ngo dul ma bor (lanyut S13). L-Ser91 utiye kabedo pa Thr in pol pa RCs mange (lanyut S13), utiye 3.8 Angstroms kud i methyl oxygen pa Q1, man ecopu mio hydrogen bonds ma nyap (lanyut 5E). Q3 nen ungo ni etiye ku tic moko, ento etiye i kabedo ma pii maru ngo ikind RC-M subunit ku LH1-α subunit 5 ku 6 (lanyut 5, D ku G). Q1, Q2 man Q3 nyo chelated quinones ma ceng ikumgi de juyubu gi in Tch. Mol, Trv. Kit 970 man Blc. Kit ma iris nen kudu (9, 10, 12) nyutho kabedo ma jugwoko ma konyo quinone i RC-LH1 complex (Figure S12G). UQs abic ma jupoko i RC-LH116 gi rwate maber ku 5.8±0.7 pa complex acel acel ma juporo ku high performance liquid chromatography (HPLC), ma UQs adek ma jupoko i RC-LH114-W gi piny ma piny pa wel ma jupimo pa 6.2±0.3 (Fig. S14) nyutho nia molecules UQ utiye ngo. ayi.
Pseudo-symmetric L man M polypeptides acel acel utiye ku TMHs abic man gi ketho heterodimer ma rwate ku BChl dimer acel, BChl monomers aryo, bacteriophage (BPh) monomers aryo, man non- Heme iron acel man UQ10 molecules acel nyo aryo. Ni kadhu kud i bedo pa hydrogen bonds iwi terminal ketone group man rwom pare ma ungeyere in Rps, carotenoids ju medo gi i M-subunit, ma julwongo cis-3,4-dehydroorhodopin. Kit lei (25). The outer membrane domain of RC-H is anchored to membrane by single TMH. Kite ma RC nen kudu urombo ku kit adek pa RC pa lei ma rwate (macalu Rba). sphaeroides (PDB ID: 3I4D). Macrocycles pa BChl ku BPh, carotenoid backbone man non-heme iron gi rwate i kind resolution range pa structures eni, calu UQ10 head group i QA site man QB quinone i RC-LH116 (Figure S15).
Bedo tiye pa RC structures aryo ku QB site occupancy rates matung tung miyo kare manyen mi ngiyo aloka loka ma rwate ku QB quinone binding. I RC-LH116 complex, QB quinone utiye i kabedo ma juketho i iye (26), ento poko pa RC-LH114-W epe ku QB quinone. QB quinone epe in RC-LH114-W, ma en e lembe mi ziazia pilembe complex ne utiye ka tic, makadhu RC-LH116 complex ku QB quinone ma juyiko maber. Kadi bende LH1 rings ario chelate quinones abusiel, abic utiye ma juyiko i RC-LH116 ring ma cego, ento adek kende re ma juyiko i RC-LH114-W ring ma oyabere. Peko maeni ma umedire copo nyutho loko RC-LH114-W QB pio pio, quinone kinetics upiyu in complex, man medo kero mi kadhu LH1 loop. Waparu niya bedu ma UQ ope i kabedo pa RC QB pa RC-LH114-W copo bedu adwogi pa piny ma tek man ma tiyo, man kabedo pa QB pa RC-LH114-W juyike pio pio i UQ turnover.
Ka QB epe, loko L-Phe217 i kabedo ma rwate ngo ku UQ10, kum ebi kelo turo ku isoprene acel mi yibe (lanyut 6A). Mi medo, aloka loka madongo ma nen kamaleng, asaga helix de (helix ma nok ikind TMH D ku E) kama L-Phe217 juloko i QB binding pocket man loko pa L-Tyr223 (lanyut 6A) Mi turo hydrogen bond ku M-Ap4 man cego binding pa QB kabedo (lanyut 6B). Helix de pivots at its base, Cα of L-Ser209 is shifted by 0.33Å, while the L-Val221Cα is shifted by 3.52Å. Aloka loka moko ope ma nen i TMH D ku E, ma copo bedu iwi dul ario zoo (lanyut 6A). Kite ma wangeyo kudu, eni ubedo gedo mir acel i RC ma juku kabedo pa QB. Poro ku kit (QB-bound) ma jik nyutho nya mapodi quinone udok piny, aloka loka mi kit ma juyubu kudu umitu emond i quinone. L-Phe217 lokore pi yubu π-stacking interaction ku quinone head group, man helix lokore woko, miyo skeleton pa L-Gly222 man side chain pa L-Tyr223 timo hydrogen bond network ku hydrogen bond structure (lanyut 6, A ku C).
(A) Cartoon ma ucungo iwi hologram (L chain, orange/M chain, magenta) man apo (gray) structure, ma i iye residues ma pire tek nyutre i ayi pa rod-like representation. UQ10 nyuthu ku rangi ma nya aceng. Lain ma jugoro ku dot nyutho hydrogen bonds ma juyubu i ot zoo. (B ku C) Nyuthu pa apolipoprotein man dul pa ring, ma nyutho side chain oxygen pa L-Phe217 ma rangi pa polo man L-Tyr223 ma rangi pa rangi makwar, kubang wadi. L subunit ubedo orange; M ku H subunits gi rangi ngo. (D ku E) Apolipoprotein (D) man (E) RC QB sites [rangi pa (A)] man Thermophilus thermophilus PSII (macol, macol ku plastic quinone; PDB ID: 3WU2) Align (58).
Ma dhanu ungiyu ngo, kadi nang dul mapol pa QB-deficient RCs manangu LH1 utiye, aloka loka ma nen i somo eni jular jukoro ngo con. Eni uketho i iye QB depletion structure kud i Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) man Rba. sphaeroides (PDB ID: 1OGV) (29), ma zo gi rom ku QB structure migi. Neno maber pa 3PRC unyutho nia LDAO (Lauryl Dimethyl Amine Oxide) detergent molecules maku i dhugola pa QB position, ma copo juku dwoko rwom pa QB. Kadi bende LDAO nyai ungo i kabedu marom i 1EYS nyo 1OGV, RCs eni juyike ku detergent marom man ecopo kelo adwogi marom. Kite ma Rba nen kudu. Sphaeroides RC co-crystallized ku cytochrome c2 (PDB ID: 1L9B) de nen calu etiye ku kabedo mi QB ma cego. Ento, i lembe maeni, N-terminal region pa RC-M polypeptide (ma timo tic ku QB binding site nikadhu kud i H bond pa Tyr residue iwi Q helix) utingo kit ma rwinyo, man aloka loka pa QB ungiyu ngo (30). Gin ma nyayu anyonga utiye nya waneno ngo kit aloka loka pa M polypeptide in RC-LH114-W structure, ma utiye marom ku N-terminal region pa RC-LH116 RC. Umitu juketh bende nia ingey jwigo LH1 antenna ma jutiyo kudu ku detergent, apolipoprotein RCs i PDB judaru, ma uketho quinone pools man lipids i kind RC ku thenge ma iye pa LH1 ring (31, 32). RC udong tiyo pilembe egwoko cofactors zo, muweko decomposable QB quinone, ma epe ku kero man pol kare rwinyo i kare mi yiko (33). Mi medo, ungeyere nia kwanyu LH1 man lipids ma rwinyo kud i RC copo bedu ku adwogi ikum tic, calu ve kwo ma nok pa charge-separated P+QB-state (31, 34, 35). Pieno, waparu niya bedo tiye pa LH1 ring ma ucungu RC copo gwoko kabedo pa QB ma "cego", man eketho kabedo ma ceng ikum QB.
Kadi bende apolipoprotein (ma epe ku QB quinone) man kite ma juyubu kudu nyuthu kende kende cal ario pa aloka loka pa kabedo pa QB, ento epe lembe mapol, lembe moko nuti ma nyutho nia mako ne copo bedu ma juketho pi juku dwoko piny pa hydroquinone. Kit tic pa quinolol ku quinone ceng ku QB site pa apolipoprotein copo bedu tung tung, ma weko RC kwero. Jubedo ka paru pi kare malac nia aloka loka mi kit ma dhanu nen kudu timo tic i mako man dwoko piny pa quinones. Ker pa RCs ma jukuboth ni dwoko piny quinones ingey loko kwo pa mudho utiye marac (36); X-ray crystallography nyutho nia ret eni utiye pi QB quinones ma juketho i "distal" conformation ma romo 4.5 Å kud i kabedo ma ceng (26) , 37). Waparu niya kit ma mako koma eni utiye lanyuth pa kite ma apolipoprotein ku dul pa dul, ma lubu dhu dhu ma kwong ku quinone man yabo pa QB site.
Type II RC ma nwangire i PSII complex pa bacteria moko ma kwo ku mac man cyanobacteria, algae man yen utiye ku yub man tic (38). Kit ma juyubu kudu piny ma nen i cal mir 6 (D ku E) nyutho bedo marom pa PSII RCs ku QB site pa bacterial RC complex. Pore ​​eni ubedo lanyuth pi kare malac pi ponjo kit ma quinone mako man dwoko piny. Lembe ma jugoro mukadho onyuthu nia aloka loka mi kite ma piny nen kudu bino ku dwoko piny pa PSII pa quinones (39, 40). Pieno, ka waparu pi gwoko kwo pa RC, yore maeni ma podi juneno ngo con copo bedu ber i kabedo pa QB pa PSII RC i lum ma oxygenated phototrophic.
Rps ΔpufW (unlabeled pufW deletion) man PufW-His (C-terminal 10x His-tagged protein-W expressed from the natural pufW locus) strains. palustris CGA009 jukoro pire i tic mwa mukadho (16). Kit maeni man isogenic wild-type parent judwok kud i friji ku ketho wel ma nok pa cells iwi PYE (ku acel acel 5 g liter -1) (ma jugwoko i LB i -80 °C, ma tye ku 50% ( w/v) glycerol) protein, yeast extract man succinate) agar [1.5%/w) plate. Juyiko sahan ne dyewor i mudho i kabedo ma ngic, man i ngeye juyike ku mac matar (~50 μmolm-2 s-1) ma jumiyo ku OSRAM 116-W halogen bulbs (RS Components, UK) pi nindu 3 ku 5 nitundu colony acel wok. Colony acel kende jutiyo kudu pi ketho 10 ml pa M22+ medium (41) ma ju medo ku 0.1% (w/v) casamino acids (ma jubi lwongo ni M22). Jupidho culture ne i the loc ma nok pa oxygen i mudho i 34°C ku yengo i 180 rpm pi sawa 48, man i ngeye 70 ml pa culture ne juyike i the loc marom pi sawa 24. Jutiyo ku semi-aerobic culture ku volume pa 1 ml pi ketho 30 ml pa M22 medium i 30 ml universal screw-top transparent glass bottle man juyiko ku agitation (~50μmolm-2 s-1) pi sawa 48 ku sterile magnetic force Stirring rod. I ngeye 30 ml pa culture juyike ku lita 1 pa culture i thenge marom, ma jutiyo kudu pi ketho lita 9 pa culture ma juyiko i ~200 μmolm-2 s-1 pi sawa 72. Jukwanyo cell ne ku centrifugation i 7132 RCF pi dakika 30, judwoko gi i ~10 ml pa 20 mM tris-HCl (pH 8.0), man jugwoko i -20°C nitundu mitire.
I ngei lyel, med deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) man Roche holoenzyme protease inhibitor tablets aryo (Merck, UK) i cell ma judwoko. I 20,000 psi French pressure cell (Aminco, USA), cell ne juturo wang 8 ku 12. I ngei wodhu cell ma rwinyo ngo man piny ma rwinyo ngo ku centrifugation i 18,500 RCF pi dakika 15 i 4°C, membrane ne juwodho kud i pigmented lysate ku centrifugation i 113,000 RCF pi sawa 2 i 43,000°C. Wek dul ma nyai man idok i 100 ku 200 ml pa 20 mM tris-HCl (pH 8.0) man ilok piny marom rom nitundo piny ma nen ungo. Membrane ma ucungo ucungo i 20 mM tris-HCl (pH 8.0) (Anatrace, USA) ma tye ku 2% (w/v) β-DDM pi sawa acel i mudho i 4°C ku jwigo moth. I ngeye, centrifuge i 70°C mi jwigo 150,000 RCF i 4°C pi sawa acel mi kwanyu piny ma nyai ungo.
Solubilizing membrane kud i ΔpufW strain ju ketho i 50 ml DEAE Sepharose ion exchange column ku column adek (CV) mi binding buffer [20 mM tris-HCl (pH 8.0) containing 0.03% (w / v) β-DDM]. Lwok column ku CV binding buffers aryo, man i ngeye lwok column ku binding buffers aryo ma tye ku 50 mM NaCl. RC-LH116 complex juwodho ku linear gradient pa 150 ku 300 mM NaCl (i binding buffer) iwi 1.75 CV, man binding complex madong juwodho ku binding buffer ma tye ku 300 mM NaCl iwi 0.5 CV. Cok spectrum mi mako piny ikind 250 ku 1000 nm, gwoko dul ma ku ratio mi mako piny (A880/A280) madit uloyu 1 i 880 ku 280 nm, dilute wang aryo i binding buffer, man ti ku yore marom kendu iwi DEAE column On purification. Dilu the fractions with A880/A280 ratios higher than 1.7 and A880/A805 ratios higher than 3.0, tim the third round of ion exchange, and retain fractions with A880/A280 ratios higher than 2.2 and A880/A805 ratios higher than 0.05. Jam piny ma juyubu eca juyike ni ~2 ml in Amicon 100,000 molecular weight cut-off (MWCO) centrifugal filter (Merck, UK), man juyike iwi Superdex 200 16/600 size exclusion column (GE Healthcare, US) ma tye ku 200 mM NaCl buffer, man juyike i buffer marom. CV. Cok spectra mi rwom pa size exclusion fraction, man iket spectra mi absorption ku A880/A280 ratios ma kadhu 2.4 man A880/A805 ratios ma kadhu 5.8 ku 100 A880, man tiyu kudgi pio pio pi yiko cryo-TEM grid nyoke gwoko i -80°C nitundo ma mitere.
Membrane ma nyayu pii kud i PufW-His juyike i 20 ml HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) ma tye ku 200 mM NaCl man 0.03% (w/w)) i IMAC buffer ( GE Healthcare). v) β-DDM]. Juyiko dul ne ku CVs abic pa IMAC buffer, man i ngeye ku CVs abic pa IMAC buffer ma tye ku 10 mM histidine. The core complex juwodho kud i column ku IMAC buffers abic ma tye ku 100 mM histidine. Thenge ma tye ku RC-LH114-W complex juyike ni ~10 ml i tank ma juyiko ku Amicon 100,000 MWCO filter (Merck, UK), juyike wang 20 ku binding buffer, man jumedo i 25 ml I DEAE Sepharose column, CVs angwen jutiyo kudu pi buffer ma jutiyo kudu i b. Lwok column ku CV binding buffers angwen, man i elute complex iwi CVs abora iwi linear gradient pa 0 ku 100 mM NaCl (i binding buffer), man CVs angwen mudong ma tye ku 100 mM binding buffer. A880/A805 ratio ma malu uloyu 4.6 juyike ni ~2 ml in Amicon 100,000 MWCO centrifugal filter, man jupongo ku 1.5 CV IMAC i wange Buffer equilibrated Superdex 200 16/600 size exclusion column, and then eluted in the same buffer over CV. Cok spectra mi mako piny pa size-exclusion fractions man iket spectra mi absorption ku A880/A280 ratios ma kadhu 2.1 man A880/A805 ratios ma kadhu 4.6 ku 100 A880, ma jutiyo kudu pio pio pi yiko grid mi TEM ma juku nyo jugwoko i -80°C nitundo ma mitire.
Leica EM GP immersion freezer jutiyo kudu pi yiko low temperature TEM grids. Jam piny ne juyike i IMAC buffer ni A880 pa 50, man 5μl juyike iwi QUANTIFOIL 1.2/1.3 carbon-coated copper mesh (Agar Scientific, UK). Keth grid i 20°C man 60% pa yamo ma rwate pi 30 s, man i thum pi 3 s, man i thum i ethane ma pii i -176°C.
Lembe pa RC-LH114-W complex jugoro iwi eBIC (Electronic Bioimaging Center) (British Diamond Light Source) ku Titan Krios microscope, ma tiyo ku kero mi 300kV, ku kero mi 130,000× man kero mi 20V. Gatan 968 GIF Quantum ku K2 peak detector jutiyo kudu pi goyo cal i yore mi kwanu lembe. Pixel ma juporo ubedo 1.048Å, man dose rate ubedo 3.83 e-Å-2s-1. Wacoko filim ne i sekond 11 man wapoko i dul 40. Ti ku kabedo ma juketho i iye carbon ni dwoko microscope, man igam filim adek i vur acel acel. I wel, filim 3130 jucoko, ku wel pa defocus ikind -1 ku -3μm.
Lembe pa RC-LH116 complex jucoko ku tic ku microscope marom i Asterbury Biostructure Laboratory (University of Leeds, UK). Lembe ne jucoko i yore mi kwano ku dit pa 130 k, man dit pa pixel juporo ni 1.065 Å ku rwom pa 4.6 e-Å-2s-1. Filim ne ju goyo i sekond 12 man jupoko i dul 48. I wel, filim 3359 jucoko, ku wel pa defocus ikind -1 ku -3μm.
Tic mi ketho lembe zo utimere i Relion 3.0 pipeline (42). Ti ku Motioncorr 2 (43) mi yiko ngwec pa mac ku dose weighting, man i ngeye ti ku CTFFIND 4.1 (44) mi nyang CTF (contrast transfer function) parameter. Fotomicrographs ma rwinyo ingey acaki mi tic eni nen i cal mir 2. S16. Template mi yero ma timo kende kende juyike ku yero 250 pixels pa 1000 particles i 250-pixel frame man no reference two-dimensional (2D) classification, ma kumeno jukwero classifications ma rombu ku sample contamination nyo ma gipe ku kit ma nen kudu. I ngeye, yero ma timo kende iwi cal ma nok zo, man RC-LH114-W ubedo 849,359 particles, man RC-LH116 complex ubedo 476,547 particles. Jam piny moko ci ma juyeru ukadhu kud i rwom aryo mi non-reference 2D classification, man ingey ngwec acel acel, jamb tic ma romo ku kabedo mi carbon, sample contamination, lembe ma nen ungo nyo jamb tic ma rwate kugi tek jukwero, ma ukelo 772,033 (90.9%) man 359,678 (75.5) particles ma jutiyo kudu pi poko 3D 3D. RC-LH114-W man RC-LH116 kubang wadi. Model mir acel mi 3D juyubu ku tic ku yore mi stochastic gradient descent. Ni tiyo ku lanyuth ma kwong, jupoko dul angwen i 3D. Ni tiyo ku lanyuth ma ni i dul eni calu lanyuth, tim 3D refining iwi particles i dul madit uloyu, man ti ku 15Å low-pass filter ma kwong ni umu kabedo pa solvent, med 6 pixels pa soft edges, man i ngeye timo pixels ni dwoko Gatan K2 peak Modulation transfer function pa top detector. Pi RC-LH114-W dataset, model ma kwong'a eni juloko ku kwanyo pek ma tek i ngete pa mask (ma jupoko kud i core complex density i UCSF Chimera). Model ma wok (resolutions pa RC-LH114-W man RC-LH116 utiye 3.91 man 4.16 Å, kubang wadi) jutiyo kudu calu lanyuth pi rwom mir aryo mi 3D classification. Jam piny ma jutiyo kudu jupoko gi i 3D class ma kwong man gipe ku winjiri ma tek ku pacu. Overlap nyo bedo mbe pa piny ma nen kamaleng. I ngei poko mir arionde mi 3D, juyeru dul ma ku resolution ma malu [Pi RC-LH114-W, dul acel utiye 377,703 particles (44.5%), pi RC-LH116, utiye dul aryo, ma wel migi romo 260,752 particles (54.77% W), kende kende ka juporo gi acel i ngeye. loko acaki ku tung tung manok]. Jam piny ma juyeru juwodho gi kendu i sanduku ma 400-pixel man juyike ku 3D refining. Solvent mask juyike ku tic ku 15Å low-pass filter, 3 pixel map expansion man 3 pixel soft mask. Ni tiyo ku per-particle CTF refinement, per-particle motion correction man per-particle CTF refinement mir aryo, 3D refinement, solvent masking man post-processing jutimo ingey step acel acel mi medu dwoko texture ma wok. Ni tiyo ku FSC (Fourier Shell Correlation Coefficient) cut-off value pa 0.143, rwom pa RC-LH114-W man RC-LH116 utiye 2.65 man 2.80Å, kubang wadi. FSC curve pa model ma tok cen nen i cal mir 2. S17.
Lembe mi protein zo juwodho kud 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) jutiyo kudu pi yubu homology model pa RC, ma uketho i iye rwom pa protein pa RC-L, RC-M man RC-H man kit pa Rba. sphaeroides RC jutiyo kudu calu lanyuth (PDB ID: 5LSE) (46). Ti ku jam tic mi "fit map" in UCSF Chimera mi kethu model ma juyubu i map (47), medu kit pa protein, man cofactor [4×BChl a (monomer library residue name = BCL), 2×BPh a (BPH), kit acel nyo aryo pa UQ10 (U10), acel non-heme iron (Fe) man acel-hexa-hexadi,44 (QAK)] ti ku Coot (48) ni medo. Calu QAK epe in monomer library, juyike ku kony pa eLBOW tool in PHENIX (49).
I ngeye, LH1 subunit juyike. I acaki, jam tic mi yiku piny ma timo kende kende i PHENIX (49) jutiyo kudu pi yiku piny moko kende kende pa LH1 ku tic ku map man LH1-α man LH1-β protein sequences calu piny ma juketho i iye. Yer LH1 subunit ma pong, kwany man iket i Coot, med sequence ma rwinyo i iye, man iketh structure ceke maber mapodi imed BCls aryo a (BCL) man spirilloxanthin (CRT) [nimakere ku Rps ma rwate The density of the LH1 complex and the known carotenoid content. Kit lei (17)]. Kwany LH1 subunit ceke, man iti ku UCSF Chimera "Docking Map Tool" mi ketho i kabedo ma ceng'ini ku non-model area pa LH1 density, man idok i Coot; dwoko timo ne wang ma LH1 subunits zo juyubu. Pi RC-LH114-W structure, ku kwanyo unallocated density in Coot, protein jupoko kud i non-protein components i USCF Chimera map man Autobuild tool jutiyo kudu pi ketho model ma kwong, man subunits madong (protein-W) Modeling. I PHENIX (49). Med lembe moko ci ma rwinyo i model ma wok in Coot (48), man idok ilok subunit ceke. Piny madong ma jupoko ngo rwate ku dul pa lipids (PDB monomer library ID pa CDL = CDL, POPC = 6PL man POPG = PGT), β-DDM detergent (LMT) man UQ10 molecules (U10). Ti ku PHENIX optimization (49) man manual optimization in Coot (48) mi yubu model ma kwong'a nitundu model statistics man nen pa piny ma rwate copo bedu maber ungo. Mi thum, ti ku LocScale (50) mi yubu map mi kabedo peri, man idok itim lembe mange mapol mi ketho rwom pa piny ma jupoko ngo man timo tic ku cing.
Peptides, cofactors man lipids man quinones mukende ma juketho i kind densities migi nen i cal 1 ku 2. S18 ku S23. Lembe mi wel pa lanyuth ma tok cen nen i Table S1.
Ka jukwanu ngo yore mukende, UV/Vis/NIR ma jucwalo jucoko iwi Cary60 spectrophotometer (Agilent, USA) i 1 nm intervals nicaku 250 nm nitundo 1000 nm man kare mi rwom pa 0.1s.
Dilu sample in quartz cuvette ku 2 mm path ni A880 pa 1, man kwany absorption spectrum ikind 400 ku 1000 nm. Dichroic spectra ma ucungo iwi Jasco 810 spectropolarimeter (Jasco, Japan) i 1 nm intervals ikind 400 nm ku 950 nm i scan rate pa 20 nm min-1.
Molar extinction coefficient juyike ku diluting core complex ni A880 ma romo 50. Dilute 10μl volume i 990μl binding buffer nyo methanol, man icok absorption spectrum ndhundhu pi jwigo BChl degradation. BChl ma nwang i sample acel acel pa methanol ju kwanu ku extinction coefficient i 771 nm pa 54.8 mM-1 cm-1, man extinction coefficient ungiyure (51). Pok wel pa BChl ma jupimo ku 32 (RC-LH114-W) nyo 36 (RC-LH116) mi nwangu wel pa core complex, ma jubi tiyo kudu pi ngeyo absorption spectrum pa sample marom ma jucoko i buffer Extinction coefficient. atira ku juwade. Pim adek ma judwoko wang mapol jutimo pi lanyuth acel acel, man rwom pa BChl Qy maximum jutiyo kudu pi kwanu. Coefficient mi tho pa RC-LH114-W ma jupimu i 878 nm utiye 3280±140 mM-1 cm-1, man coefficient mi tho pa RC-LH116 ma jupimu i 880 nm utiye 3800±30 mM-1 cm-1.
UQ10 jupimu nimakere ku yore ma nwangire i (52). I lembe ma nok, reverse phase HPLC (RP-HPLC) jutimo ku kony pa Agilent 1200 HPLC system. Lar 0.02 nmol pa RC-LH116 nyo RC-LH114-W i 50μl pa 50:50 methanol:chloroform ma tye ku 0.02% (w/v) ferric chloride, man i med pre-equilibrated Beckman Coulter Ultrasphere ODS 4 mml i 1-1-1-min. 40°C in HPLC solvent (80:20 methanol:2-propanol) iwi ×25 cm column. Tim isocratic elution in HPLC solvent ni neno absorbance i 275 nm (UQ10), 450 nm (carotenoids) man 780 nm (BChl) pi sawa acel. Ma malu in 275 nm chromatogram i dakika 25.5 juyike, ma epe ku piny mukende ma jucopu nwangu. Kabedo ma juketho i iye jutiyo kudu pi kwanu wel molar pa UQ10 majuwodho ku nen pa calibration curve majukwanu kud i injection pa pure standards nicaku 0 ku 5.8 nmol (Figure S14). Lanyuth acel acel jutimo sayusac iwie wang adek, man lembe ma jukoro urombo ku SD pa wel.
Yath ma tingo RC-LH1 ku rwom ma malu pa Qy 0.1 juyike ku 30 μM ma judwoko piny pa adunde pa thwol cytochrome c2 (Merck, UK) man 0 ku 50 μMUQ2 (Merck, UK). Juyiko lanyuth adek mi 1-ml i rwom pa UQ2 acel acel man juyike dyewor i mudho i 4°C mi neno ni gicokiri maber i mudho mapodi jupimo ngo. Adwogi ne jubolo i OLIS RSM1000 modular spectrophotometer ma juketho i iye 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm middle man 0.6 mm outlet slits. Filter ma bor pare 600 nm juketho i dhugola pa sample phototube man reference photomultiplier tube pi jwigo ler ma nyai. Absorbance jubedu ka lubo i 550 nm ku kare mi 0.15 s. Lero ma wok kud i 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) nikadhu kud i fiber optic cable ma tek pa 90% nikadhu kud i DC2200 controller (Thorlabs Ltd., UK) man wok kud i kama ler uwok kud i angle pa 90°. Jam pimo ubedo tung tung ku kiyo pi dwoko lero moku ci ma jular unwangu ngo ku lanyuth. Nen rwom pa pii 10 s mapodi ler pa 50 s ukadhu. I ngeye, jumedo lubo tok rwom pa pii pi 60 s i mudho pi ni neno ka quinolol dwoko piny cytochrome c23 + (nen cal S8 pi lembe ma nyen).
Lembe ne jutiyo kudu ku ketho rwom ma lageng ikind 0.5 ku 10 s (nimakere ku rwom pa UQ2) man rwom pa lanyuth adek zoo i rwom pa UQ2 acel acel. RC-LH1 ma ju kwanu ku extinction coefficient acel acel jutiyo kudu pi loko rate ni catalytic efficiency, ma jugoro i Origin Pro 2019 (OriginLab, USA), man juporo ku Michaelis-Menten model pi ni nyang Km man Kcat values.
Pi pimo rwom ma lac, RC-LH1 sample juyike ni ~2μM in IMAC buffer ma tye ku 50 mM sodium ascorbate (Merck, USA) man 0.4 mM Terbutin (Merck, USA). Ascorbic acid jutiyu kudu calu electron donor, man tert-butaclofen jutiyo kudu calu QB inhibitor mi neno ni RC donor madit udong ma nok (niwacu, epe ku photooxidized) ikind kare mi pimo. Maromo 3 ml pa lanyuth jumedo i cell ma lokore (ma romo 0.1 m in diameter, 350 RPM) ku bor pa 2 mm mi yore mi neno ni lanyuth ma ni i yore mi laser utiye ku kare ma uketh pi timo piny macol ikind pulses mi nyayu lyethu. Ti ku ~100-fs laser pulses mi medo kero pa Ti: Sapphire laser system (Spectra Physics, USA) mi nyayu kero pa sample i 880 nm i rwom mi 1 kHz (20 nJ pi NIR nyo 100 nJ pi Vis). Mapodi icoko lembe, keth lanyuth ne i mac ma nyai pi dakika 30. Nyuthu piny bi kelo inactivation pa QA (copu bedu dwoko piny QA wang acel nyo aryo). Ento nen nia lembe eni copo dwoko cen, kum ingey kare malac mi timo lembe macol, RC bidok cen moth moth i tic pa QA. Helios spectrometer (Ultrafast Systems, USA) jutiyo kudu pi pimo transient spectra ku kare mi -10 ku 7000 ps. Ti ku Surface Xplorer software (Ultrafast Systems, USA) mi poko lembe ma jugwoku, man idok i rwom acel. Ti ku CarpetView software package (Light Conversion Ltd., Lithuania) ni tiyo ku lembe ma jucoko pi nwangu spectra ma tung tung ma mako rwinyo, nyo ti ku tic ma ketho exponents mapol ku dwokowang pa jam tic ni rombu ku single-wavelength spectral evolution in Origin (OriginLab, USA).
Calu ma juweco pire malu (53), juyike filim mi photosynthetic ma tye ku LH1 complex ma epe ku RC man peripheral LH2 antenna. Membrane ne juyike i 20 mM tris (pH 8.0) man juyike i quartz cuvette ku 2 mm optical path. Laser pulse pa 30nJ jutiyo kudu pi nyayu anyonga i 540 nm ku kare mi -10 ku 7000 ps. Ti ku lembe ma jukwanu calu ma juloko pi Rps. lanyuth pa wadi.
Membrane ne jupoko ku centrifugation i 150,000 RCF pi sawa 2 i 4°C, man i ngeye absorbance pare i 880 nm judwoko i 20 mM tris-HCl (pH 8.0) man 200 mM NaCl. Lar membrane ni 2% (w/v) β-DDM pi sawa acel i mudho i 4°C. Lanyuth ne juyike i 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) ni 2.5 mg ml-1 (Bio-Rad analysis). Tic mukende jutimo ku yore ma juwodho con (54), nicaku ku nyayu 50 μg pa protein i 50 μl TEAB ma tye ku 1% (w/v) sodium laurate (Merck, UK). I ngei sonication pi 60 s, judwoko piny ku 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) i 37°C pi dakika 30. Pi S-alkylation, jwigo lanyuth ku 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) man imed kud i 200 mM isopropanol stock solution pi dakika 10 i ot. Nwangu piny ma nyayu remo jutimo ku medo 2 μg trypsin/endoproteinase Lys-C mixture (Promega UK) man juyike i 37°C pi sawa adek. Laurate surfactant juwodho ku medu 50 μl ethyl acetate man 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) man vortexing pi 60 s. Poko pa dul ne jumedo ku centrifugation i 15,700 RCF pi dakika abic. Nimakere ku cik pa ju ma yiku piny, C18 spin column (Thermo Fisher Scientific, UK) jutiyo kudu pi jwigo man jwigo kadu piny ma piny ma tingo peptide. I ngei jwigo ku vacuum centrifugation, jupoko lanyuth ne i 0.5% TFA man 3% acetonitrile, man 500 ng juporo ku nanoflow RP chromatography man mass spectrometry nitio ku lembe ma jukoro con.
Ti ku MaxQuant v.1.5.3.30 (56) pi ngeyo man pimo wel pa protein pi sayu Rps. palustris proteome database (www.uniprot.org/proteomes/UP000001426). Lembe pa mass spectrometry proteomics ju ketho i ProteomeXchange Alliance nikadhu kud i PRIDE partner repository (http://proteomecentral.proteomexchange.org) ithe dataset identifier PXD020402.
Pi nyang ikum RPLC ma rwate ku electrospray ionization mass spectrometry, RC-LH1 complex juyike kud i wild-type Rps. Ni tiyo ku yore ma jugoro con (16), dwong pa protein ma juyubu i palustris cells ubedo 2 mg ml-1 in 20 mM Hepes (pH 7.8), 100 mM NaCl man 0.03% (w/v) β- (Bio-Rad analysis) ) DDM. Nimakere ku cik pa ju ma yiku piny, ti ku 2D purification kit (GE Healthcare, USA) mi kwanyu 10 μg pa protein ku yore mi pii, man ilok piny ne i 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile man 20% (v/v) pii. Microliter abic juporo ku RPLC (Dionex RSLC) man mass spectrometry (Maxis UHR-TOF, Bruker). Ti ku MabPac 1.2×100 mm column (Thermo Fisher Scientific, UK) pi poko i 60°C man 100μlmin -1, ku gradient pa 85% (v / v) solvent A [0.1% (v / v) FA man 0.02% (V/v) TFA aqueous solution] ni 85% Bv/v) [0.1%(v/v) FA man 0.02%(v/v) in 90%(v/v) acetonitrile TFA] Ni tiyo ku electrospray ionization source man default parameters pi dakika ma kadhu 60, mass spectrometer nwangu 100 ku 2750 m/z (mass-to-charge ratio). Ku kony pa ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), ket mass spectrum ni subunits mi complex.
Jupidho adila pi sawa 72 i the 100 ml NF-low (10μMm-2 s-1), medium (30μMm-2 s-1) nyo high (300μMm-2 s-1) light. M22 medium (M22 medium ma ammonium sulfate juweko man sodium succinate juloko ku sodium acetate) i 100 ml screw-top bottle (23). I kind 30-s abic, 0.1 micron glass beads juyike i volume ratio pa 1:1 mi poko cell man juyike iwi ice pi dakika 5. Piny ma nyai ungo, cell ma nyai man jamb moko ma nyai juwodho gi ku centrifugation i 16,000 RCF pi dakika 10 i benchtop microcentrifuge. Membrane ne jupoko i Ti 70.1 rotor ku 100,000 RCF i 20 mM tris-HCl (pH 8.0) ku 40/15% (w/w) sucrose gradient pi sawa 10.
Calu ma jukoro i tic mwa mukadho, immunodetection pa His tag iwi PufW (16). I lembe ma nok, core complex ma juyiko (11.8 nM) nyo membrane ma tye ku RC marom (ma juyiko ku oxidation ma juwodho spectrum ma judwoko piny man ma juporo ku pek pa gel ma juyiko) i 2x SDS loading buffer (Merck, UK) ma juyiko wang aryo. Jam piny ne jupoko iwi 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). Gel moko juyike ku Coomassie Brilliant Blue (Bio-Rad, UK) mi ketho man nen RC-L subunit. Protein ma ni iwi gel mir aryo juwodho i methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) pi immunoassay. PVDF membrane ubedo blocked in 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 man 5% (w / v) skimmed milk powder, man i ngeye juyike ku anti-His primary antibody (i Dilute the antibody buffer [pH 7.6 7.6), 150 mM NaCl man 0.05% (v/v) Tween-20] in 1:1000 A190-114A, Bethyl Laboratories, USA) pi sawa angwen. I ngei lwoko wang 3 pi dakika abic i antibody buffer, membrane ne juporo ku horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (diluted 1:10,000 in antibody buffer) Incubate to allow detection (5 minutes after washes 3 in antibody buffer) using WAR EST 2000. chemiluminescence substrate (Cyanagen, Italy) man Amersham Imager 600 (GE Healthcare, UK).
Ni goyo apoka poka pa kero pa gel acel acel nyo immunoassay lane, medo kabedo ma piny pa peak man kwanu kero pa RC-L (gel ma rangi) man Protein-W (immunoassay), i ImageJ (57) Tiyu ku cal. Ratio eni juloko gi ni molar ratio ni kadhu nia ratio pa RC-L ku protein-W i RC-LH114-W ma leng ubedo 1:1 man ni dwoko lembe ceke ni bedo marom.
Pi lembe mukende ma medo ikum lembe maeni, 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, C. Necoil, Necoil Hunter.
Kit ma light trap 1 complex utiye kudu i reaction center mio ngec manyen ikum 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, C. Necoil, Necoil Hunter.
Kit ma light trap 1 complex utiye kudu i reaction center mio ngec manyen ikum quinone dynamics.
©2021 Kanica mi Amerika pi dongo ngec mi ngec. twero ceke jugwoko. AAAS ubedo wadi pa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef man COUNTER. ScienceAdvances ISSN 2375-2548.