Adreesị ugbu a: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Ụlọ Ọrụ Nyocha Ihe Ndị Dị Ndụ Elektrọnik.
Ihe mgbagwoju anya nke na-ewe ihe ubi nke ìhè nke etiti mmeghachi omume 1 (RC-LH1) bụ isi ihe mejupụtara photosynthesis nke nje bacteria na-acha odo odo. Anyị webatara usoro microscopy cryo-electron abụọ nke mgbagwoju anya RC-LH1 site na Rhodopseudomonas palustris. Nhazi mkpebi 2.65-Å nke mgbagwoju anya RC-LH1 nwere loops LH1 iri na anọ gbara RC gburugburu, nke protein W na-akwụsị, ebe mgbagwoju anya na-enweghị protein-W bụ ihe mejupụtara RC kpamkpam nke RC gbara gburugburu. Emechiri loop LH1 nkeji iri na isii. Ntụnyere nke usoro ndị a na-enye nghọta banyere ike nke quinone na mgbagwoju anya RC-LH1, gụnyere mgbanwe nhazi a na-achọpụtabeghị na mbụ mgbe ejiri quinone na saịtị RC QB, yana ebe ebe njikọ quinone enyemaka dị, nke na-enyere aka ịgafe ha na RC. Nhazi pụrụ iche nke protein W na-egbochi mmechi nke loop LH1, si otú a na-emepụta ọwa maka ime ka mgbanwe quinone/quinolone dị ngwa ngwa.
Ike nke photosynthesis na-enye nwere ike ịkwado ihe fọrọ nke nta ka ọ bụrụ ndụ niile dị n'ụwa, o nwekwara nnukwu ikike maka teknụzụ anyanwụ. Ọ bụ ezie na ọ na-akwalite photosynthesis zuru ụwa ọnụ, nje bacteria na-acha odo odo na-egosipụtakwa ụdị ike dị iche iche na ikike metabolic. Ha nwere ike izere photosynthesis ma too ka nje bacteria heterotrophic n'ọchịchịrị, nwere ike idozi nitrogen na carbon dioxide, mepụta hydrogen, ma mebie ihe ndị na-esi ísì ụtọ (1-3). Iji nye ike maka usoro ndị a, a ga-agbanwe ìhè ngwa ngwa na nke ọma ka ọ bụrụ ike kemịkal. Usoro a na-amalite mgbe mgbagwoju anya antenna na-ejide ìhè na-amịkọrọ ìhè ma na-ebufe ike ahụ na ebe mmeghachi omume (RC), si otú a na-amalite nkewa chajị (4-7). Isi ihe dị na photosynthesis na nje bacteria na-acha odo odo bụ ụdị RC nke 2, gbara ya gburugburu site na mgbagwoju anya na-ewe ihe ubi 1 (LH1), na-emepụta mgbagwoju anya RC-LH1. A na-emepụta LH1 site na ọtụtụ heterodimers αβ gbagọrọ agbagọ, nke ọ bụla n'ime ha na-ejikọta chlorophyll bacteria abụọ (BChl) otu molekul na otu ma ọ bụ carotenoids abụọ (8-12). Anteena LH1 kachasị mfe nwere heterodimers αβ 16 ma ọ bụ 17 gbara RC (9-13) gburugburu n'ime mkpọchi mechiri emechi, mana na mgbagwoju anya ndị ọzọ, peptides transmembrane na-egbochi ọganihu nke LH1 gbara ya gburugburu, si otú a na-akwalite mgbasa quinol/quinone n'etiti RC na cytochrome bc1 complex (11, 13-15). Osisi fototrophic odo odo Rhodopseudomonas (Rps.) bụ ihe nlereanya nke nwere ike ịghọta ike na nnyefe eletrọn nke na-akwado photosynthesis. Nhazi kristal mbụ nke Rps. Ihe nlereanya nke mgbagwoju anya palustris RC-LH1 bụ RC, gbara ya gburugburu site na loops 15 heterodimeric LH1, nke protein a na-amaghị aha ya bụ "Protein W" (14) na-akwụsị. Emesịa achọpụtara Protein-W dị ka RPA4402, nke bụ protein 10.5kDa nke a na-akọwaghị nke ọma yana helices transmembrane atọ a buru amụma (TMH) (16). Anyị na-atụ aro ka a gbanwee aha mkpụrụ ndụ ihe nketa rpa4402 nke na-agbanwe protein W ka ọ bụrụ pufW ka o kwekọọ na aha e ji mee mkpụrụ ndụ ihe nketa nke na-agbanwe RC-L, M (pufL, pufM) na LH1α, β (pufA, pufB). Ọ dị mma ịmara na protein-W dị naanị n'ihe dị ka 10% nke RC-LH1, na-ekpughe Rps. palustris na-emepụta ihe mgbagwoju anya RC-LH1 abụọ dị iche iche. N'ebe a, anyị na-akọ akụkọ nhazi cryo-EM (cryo-EM) dị elu nke mgbagwoju anya abụọ dị mkpa, otu nwere protein W na 14 αβ heterodimers, nke ọzọ na-enweghị protein W na mkpọchi 16 Heterodimer LH1 mechiri emechi. Nhazi anyị na-anọchite anya mgbanwe nzọụkwụ na nghọta nke mgbagwoju anya RC-LH1 nke Rps. palustris, n'ihi na anyị enyochala ọnụọgụgụ ndị yiri nke ọ bụla ma nwee mkpebi zuru oke iji kenye peptide na pigments jikọtara ọnụ na lipids na quinones metụtara ya nke ọma. Ntụnyere nke usoro ndị a na-egosi na protein TMH atọ-W nke a na-ahụbeghị na mgbagwoju anya RC-LH1 ọ bụla ọzọ ruo ugbu a na-emepụta ọwa quinone iji mee ka mgbanwe quinone/quinolone dịkwuo ngwa ngwa. Achọpụtala ọtụtụ ebe njikọta lipid na quinone echekwara, anyị ekpughewokwa mgbanwe nhazi ọhụrụ mgbe njikọta nke quinone na RC gasịrị, nke nwere ike ịdị mma maka photosystem II (PSII) RC nke ihe ndị dị ndụ na-enweta oxygen. Nchọpụta anyị na-enye nghọta ọhụrụ banyere kinetics nke njikọta quinone/quinolone na mgbanwe na RC-LH1 isi complex nke nje bacteria na-acha odo odo phototrophic.
Iji mee ka ọmụmụ ihe zuru ezu nke mgbagwoju anya abụọ dị na Rps. palustris dị mfe, anyị na-ekewapụ RC-LH1 ọ bụla site na usoro kemịkalụ. E sachara mgbagwoju anya nke na-enweghị protein W (nke a na-akpọ ΔpufW) site na nje nke na-enweghị mkpụrụ ndụ ihe nketa pufW (16), naanị otu mgbagwoju anya RC-LH1 ka enwere ike ịmepụta. A na-emepụta mgbagwoju anya nke nwere protein W site na nje. A na-agbanwe protein W nke ụdị a site na iji akara His 10x na C-terminus ya, ka e wee nwee ike ijikọta mgbagwoju anya nke nwere protein W nke ọma na ọtụtụ protein W na-enweghị site na ikpuchi ígwè. A na-ekewa mgbagwoju anya ahụ nke ọma (16) Affinity Chromatography (IMAC).
Dịka egosiri na Foto 1, ihe mgbagwoju anya abụọ a nwere obere RC atọ (RC-L, RC-M na RC-H) nke e ji eriri LH1 gbaa gburugburu. Nhazi 2.80-A nke ihe mgbagwoju anya na-enweghị protein-W na-egosi heterodimers 16 αβ, na-emepụta okirikiri LH1 mechiri emechi gburugburu RC, nke a na-akpọzi RC-LH116 complex. Nhazi 2.65Å nke ihe mgbagwoju anya nwere protein-W nwere LH1 14-heterodimer nke protein-W kwụsịrị, nke a na-akpọzi RC-LH114-W.
(A na B) Nnọchianya elu nke ihe mejupụtara ahụ. (C na D) Agba ndị ejikọtara ọnụ nke a na-egosipụta n'ogwe osisi. (E na F) Ihe mgbagwoju anya ndị a hụrụ site na elu cytoplasmic nwere peptides na obere LH1 nke a na-anọchite anya na katọn, a na-agụkwa ha n'aka elekere site na oghere protein-W [nke kwekọrọ na ọnụọgụ Rba. sphaeroides complex (13)]. Maka LH1-α, agba nke obere protein bụ odo odo; maka LH1-β, agba nke obere protein bụ acha anụnụ anụnụ; maka protein-W, protein bụ ọbara ọbara; maka RC-H, ọ bụ cyan; maka RC-L, ọ bụ oroma; maka RC-M, magenta. A na-anọchite anya cofactors site na mkpara, akwụkwọ ndụ akwụkwọ ndụ na-anọchite anya molekul BChl na BPh a, odo odo na-anọchite anya carotenoids, odo na-anọchitekwa molekul UQ10. (G na H) Echiche buru ibu nke oghere protein-W na mpaghara nhata nke mgbagwoju RC-LH114-W (G) na mgbagwoju RC-LH116 (H). A na-egosi ihe ndị na-eme ka oghere dị n'ime ya n'ụdị ihe e ji emeju oghere, a na-egosikwa quinone chelated na acha anụnụ anụnụ. A na-egosi oghere protein-W site na ahịrị acha anụnụ anụnụ na (G), a na-egosipụtakwa obere oghere ebe quinone/quinolol na-agbasa na mgbanaka LH116 site na ahịrị ojii nwere akara na (H).
Foto nke 1 (A na B) na-egosi RC gbara ya gburugburu site na usoro LH1αβ heterodimers mepere emepe ma ọ bụ mechiri emechi, nke ọ bụla na-ejikọta BChl abụọ na otu carotenoid (Foto nke 1, C na D). Ọmụmụ ihe ndị gara aga egosila na Rps bụ mgbagwoju anya LH1. N'ụzọ biosynthetic nke spirulina xanthin, ụdị ndị a nwere ọtụtụ carotenoids dị iche iche (17). Agbanyeghị, spiropyrroxanthin bụ carotenoid kachasị na njupụta ya dị mma. Ya mere, anyị họọrọ ịmepụta ụdị spiroxanthin na ebe njikọ LH1 niile. Alfa na beta polypeptides bụ otu TMH nwere mpaghara mpụta membrane dị mkpụmkpụ (Foto nke 1, A, B, E, na F). Ọ bụ ezie na ahụghị njupụta nke residues 17 na C-terminus, e kewara alpha polypeptide site na Met1 ruo Ala46 na mgbagwoju anya abụọ ahụ. E belatara β polypeptide site na Gly4 ruo Tyr52 na RC-LH116, site na Ser5 ruo Tyr52 na RC-LH114-W. A hụghị njupụta nke ihe fọdụrụ n'ime N-terminal 3 ma ọ bụ 4 ma ọ bụ C-terminal 13 (Foto S1). Nyocha nke oke spectrometry nke mgbagwoju anya RC-LH1 agwakọtara nke e si n'ụdị anụ ọhịa ahụ pụta gosiri na mpaghara ahụ na-efu bụ nsonaazụ nkewa heterologous nke peptides ndị a (Foto S1 na S2). A hụkwara nhazi N-terminal nke α-Met1 (f). Nyocha ahụ gosiri na α-peptide nwere ihe fọdụrụ fMet1 ruo Asp42/Ala46/Ala47/Ala50, β-peptide nwekwara ihe fọdụrụ n'ime Ser2 ruo Ala53, nke kwekọrọ na map njupụta EM dị ala.
Nhazi nke α-His29 na β-His36 na-eme BChls ihu na ihu; αβ heterodimer ọ bụla na-ezukọta na ndị agbata obi ya iji mepụta oghere mepere emepe (RC-LH114-W) ma ọ bụ mkpọchi mechiri emechi (RC-LH116) gburugburu RC. Usoro pigment ejikọtara exciton (Foto 1, C na D). Ma e jiri ya tụnyere eriri 877 nm nke RC-LH114-W, mgbanwe ọbara ọbara nke RC-LH116 880 nm bụ 3 nm (Foto 2A). Agbanyeghị, spectrum dichroism okirikiri fọrọ nke nta ka ọ bụrụ otu (Foto 2B), na-egosi na ọ bụ ezie na enwere ọdịiche doro anya n'etiti oghere mepere emepe na nke mechiri emechi, gburugburu ebe obibi nke BChls yiri nnọọ. Mgbanwe ọbara ọbara nke absorption nwere ike ịbụ nsonaazụ nke mbelata mmegharị okpomọkụ na mmụba kwụsie ike na loop mechiri emechi (18, 19), mgbanwe na njikọ pigment nke loop mechiri emechi kpatara (20, 21), ma ọ bụ njikọta nke mmetụta abụọ a (11).
(A) Ultraviolet/a na-ahụ anya/n'ihe dị nso na infrared absorption spectrum, nke e ji agba ha kwekọọ akara ma mee ka ha dị mma ruo na BPh elu na 775 nm. (B) Ọdịdị dichroism okirikiri nke a na-eme ka ọ dị mma ruo na BChl absorbance na 805 nm. (C na D) Ụdị ΔA ahọpụtara site na ụdị RC-LH114-W complex (C) na RC-LH116 complex (D) nke a na-edozi oge. Maka ntụnyere ka mma, a na-eme ka ụdị spectra niile bụrụ nke a na-ahazi ruo ∆A nke −A na 0.2 ps. (E) Ọnụego nke oxidation cytochrome c2 mgbe e mechara radieshon n'ihu ọtụtụ njupụta nke UQ2 (lee Foto S8 maka data raw). (F) Na mkpụrụ ndụ ndị a zụlitere n'okpuru obere, ọkara ma ọ bụ nnukwu ọkụ (10, 30 ma ọ bụ 300μMm-2 s-1, n'otu n'otu), protein W na RC-L subunits na ngwakọta dị ọcha na oke akpụkpọ ahụ kewapụrụ. Chọpụta ọkwa protein site na electrophoresis gel SDS-polyacrylamide na immunoassay (lee Foto S9 maka data raw). Chọpụta oke ya ma e jiri ya tụnyere mgbagwoju anya RC-LH114-W dị ọcha. Oke stoichiometric nke RC-L na protein-W nke mgbagwoju anya bụ 1:1.
BChls nọ n'ọnọdụ 1 n'ime oghere αβ14 nke RC-LH114-W nke gbanwere agbanwe (Foto 1, A, C, na E) dị nso na onye na-enye RC mbụ (P) site na 6.8Å karịa BChls ndị yiri ya na RC-LH116 (Foto 1, B, D, na F, na Foto S3); agbanyeghị, kinetics absorption nke mgbagwoju anya abụọ ahụ na-egosi na maka RC-LH114-W na RC-LH116, oge nnyefe ike mkpali site na LH1 ruo RC bụ 40 ±4 na 44±3 ps (Foto 2). , C na D, Foto S4 na Tebụl S2). Enweghịkwa nnukwu ọdịiche dị na mbufe eletrọniki n'ime RC (Foto S5 na ederede mgbakwunye metụtara ya). Anyị na-enyo enyo na nkwekọ nke oge mbufe ike n'etiti LH1 na RC-P bụ n'ihi anya, akụkụ na ike nwere ike nke ọtụtụ BChl na loops LH1 abụọ ahụ. O yiri ka inyocha usoro ike LH1 iji ruo ebe kacha nta adịghị ngwa ngwa karịa mbufe ike ozugbo site na ebe ndị na-adịghị mma gaa na RC. Oghere LH1 mepere emepe na RC-LH114-W nwekwara ike ịgafe mmegharị okpomọkụ dị obere n'okpuru ọnọdụ okpomọkụ dị ala maka nyocha nhazi, enwerekwa nhazi mgbanaka αβ14 dị ogologo na okpomọkụ ụlọ site na anya agba nke βBChls na ọnọdụ RC 1.
Ihe mejupụtara RC-LH116 nwere 32 BChls na carotenoids 16, nhazi ya n'ozuzu ya bụ otu ihe ahụ e si na Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 ụdị (PDB ID 7C9R) (12) na algae akwụkwọ ndụ akwụkwọ ndụ (Blc.viridis) (PDB ID 6ET5) (10). Mgbe nhazi ahụ gasịrị, naanị obere mgbanwe dị na ọnọdụ nke αβ heterodimers ka a hụrụ, ọkachasị 1-5, 15, na 16 (Foto S6). Ọnụnọ nke protein-W nwere mmetụta dị ukwuu na nhazi nke LH1. A na-ejikọ TMHs atọ ya site na obere loops, yana N-terminal n'akụkụ lumen nke mgbagwoju anya na C-terminal n'akụkụ cytoplasmic (Foto 1A na 3, A ruo D). Protein-W bụ hydrophobic (Foto 3B), TMH2 na TMH3 na-akpakọrịta na LH1αβ-14 iji mepụta elu transmembrane (Foto 3, B na E ruo G). Njikọ ahụ bụ isi ihe mejupụtara Phe, Leu na Val residues na mpaghara transmembrane. A na-ejikọta ihe fọdụrụ ndị a na amino acid hydrophobic na pigments αβ-14. Ụfọdụ residues polar na-enyekwa aka na mmekọrịta ahụ, gụnyere njikọ hydrogen dị n'etiti W-Thr68 na β-Trp42 n'elu oghere dị mgbagwoju anya (Foto 3, F na G). N'elu cytoplasm, Gln34 dị n'akụkụ otu keto nke carotenoids αβ-14. Na mgbakwunye, edoziri molekul n-dodecyl β-d-maltoside (β-DDM), ọdụ ya hydrophobic gbasaa ruo na njikọ dị n'etiti protein-W na αβ-14, ọdụ lipid nwekwara ike ịnọ n'ahụ. Anyị hụkwara na mpaghara mkpebi C-terminal nke protein W na RCH dị nnọọ nso, mana ọ bụghị n'ime oke nke ịmepụta mmekọrịta kpọmkwem (Foto 1, A na E). Agbanyeghị, enwere ike inwe mmekọrịta na amino acid C-terminal nke protein abụọ a na-edozighị, nke nwere ike inye usoro maka ịchịkọta protein-W n'oge njikọta nke mgbagwoju anya RC-LH114-W.
(A) Protein-W, nke na-eche ihu na njikọ ya na LH1αβ14 n'ụdị katuunu, nwere agbụ akụkụ dị ka mkpanaka (uhie), nke egosiri n'akụkụ eserese ike electrostatic (elu isi awọ doro anya nwere ọkwa nhazi nke 0.13). (B) Protein-W nke elu agba hydrophobic na-anọchi anya ya. A na-egosi mpaghara polar na charge na cyan, a na-egosi mpaghara hydrophobic na ọcha, a na-egosikwa mpaghara hydrophobic siri ike na oroma. (C na D) Protein-W nke egosiri na katuunu, nhazi ya bụ otu ihe ahụ dị na (A) (C), ma na-atụgharị ya site na 180° (D). Dịka ọnọdụ dị na usoro ahụ si dị, ihe fọdụrụ a na-ahụkarị na-eji usoro agba egwurugwu eme ihe, ebe N-terminal bụ acha anụnụ anụnụ na C-terminal bụ uhie. (E) Protein-W n'otu echiche ahụ dị na (A), na ihe fọdụrụ na njikọ nke protein-W:LH1 bụ site na mkpara nwere akara agbakwunyere. (F) A na-atụgharị Protein-W 90° ma e jiri ya tụnyere (E) na LH1αβ14 na ihe nnọchianya katọn, ma e jiri ya tụnyere ihe fọdụrụ n'ihu na ihe nnọchiteanya mmanya ahụ. A na-akpọ ihe fọdụrụ n'elu beta polypeptide akara. A na-egosi cofactor dị ka ogwe kwekọrọ na agba nke Foto 1, a na-egosi β-DDM gbawara agbawa na isi awọ, a na-egosikwa oxygen na ọbara ọbara. (G) A na-atụgharị echiche dị na (F) 180°, yana ihe fọdụrụ pụtara ìhè nke alpha polypeptide a kpọrọ aha.
Protein-W na-anọchi αβ heterodimer (nke 15 na Foto 1F), si otú a na-egbochi mmechi loop na ịtụgharị heterodimers atọ mbụ αβ. A hụrụ na akụkụ ntụgharị kachasị nke heterodimer αβ-1 mbụ ma e jiri ya tụnyere ihe nkiri nkịtị bụ 25° ruo 29° (Foto 1, A na E), nke e guzobere site na ntụgharị 2° ruo 8° nke αβ-1 na RC A dị nkọ-LH116 (Foto 1, B na F). Heterodimers nke abụọ na nke atọ na-agbada na 12° ruo 22° na 5° ruo 10°, n'otu n'otu. N'ihi mgbochi steric nke RC, ntụgharị nke αβ-1 anaghị agụnye ụzọ nke abụọ nke αβ (nke kwekọrọ na αβ nke 16 na Foto 1F), si otú a na-emepụta oghere doro anya na mgbanaka LH1 (Foto 1, A na E). N'ihi enweghị heterodimers αβ abụọ, ya na mfu nke BChl anọ na carotenoids abụọ, ọ dịghị nke carotenoids ọ bụla na-ejikọ na subunit αβ-1 gbagọrọ agbagọ, nke na-eme ka mgbanaka LH114-W nwere carotenoids 13. Ndị anaghị eri anụ na 28 BChls. Atụmatụ mkpebi mpaghara nke mgbagwoju anya abụọ dị na mpaghara αβ1 ruo 7 dị ala karịa nke ndị ọzọ nke LH1 loop, nke nwere ike igosipụta plasticity dị n'ime nke subunit LH1 dị n'akụkụ saịtị RC QB (Foto 4).
E gosiri foto nke RC-LH114-W (A na B) na RC-LH116 (C na D) site n'otu echiche elu/akụkụ (A na B) (A na C) na elu oghere nke Fig. 1. (B na D). E gosiri igodo agba n'aka nri.
Naanị ihe mgbagwoju anya isi ihe dị iche iche nwere oke stoichiometric nke 1:14 bụ Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Agbanyeghị, protein W na PufX enweghị njikọ doro anya, ha nwekwara mmetụta dị ukwuu na nhazi LH1 ha. PufX bụ otu TMH nwere ngalaba cytoplasmic N-terminal nke na-akpakọrịta na akụkụ cytoplasmic nke subunit RC-H (13) n'ọnọdụ kwekọrọ na Rps. palustris LH116αβ-16. PufX na-emepụta ọwa maka mgbanwe quinone/quinolone n'etiti RC-LH1 na cytochrome bcl complex ma dị na Rba. sphaeroides core complex niile (13). Ọ bụ ezie na njikọ monomer-monomer dị na Rba. Sphaeroides RC-LH1-PufX dimer dị n'ọnọdụ njikọ nke protein W na RC-LH114-W, oghere nke PufX na protein-W kpalitere dịkwa n'ọnọdụ nhata (Foto S7A). Oghere dị na RC-LH114-W na-adakọkwa na ọwa quinone echiche (8) nke Pseudomonas rosea LH1, nke peptides na-anaghị ejikọta na protein W ma ọ bụ PufX na-emepụta (Foto S7B). Na mgbakwunye, ọwa quinone dị na Blc. Emerald green LH1 nke e guzobere site na iwepụ otu γ subunit (7) dị n'ọnọdụ yiri ya (Foto S7C). Ọ bụ ezie na protein dị iche iche na-eme ya, ọdịdị nke ọwa quinone/quinolol ndị a n'ọnọdụ nkịtị na mgbagwoju anya RC-LH1 yiri ka ọ bụ ihe atụ nke mgbanwe mgbanwe, na-egosi na oghere nke protein W kere nwere ike ịrụ ọrụ dị ka ọwa quinone.
Oghere dị na loop LH114-W na-enye ohere ịmepụta mpaghara membrane na-aga n'ihu n'etiti oghere dị n'ime nke mgbagwoju anya RC-LH114-W na akpụkpọ ahụ buru ibu (Foto 1G), kama ijikọ ngalaba abụọ ahụ site na oghere protein dịka ọ dị na protein. Mgbakwụnye RC-LH116 yiri mgbagwoju anya Tch. dị ka agịga (22) (Foto 1H). Ebe ọ bụ na mgbasa nke quinone site na akpụkpọ ahụ dị ngwa karịa mgbasa site na ọwa protein dị warara, loop LH114-W mepere emepe nwere ike ikwe ka ntụgharị RC dị ngwa karịa loop LH116 mechiri emechi, mgbasa nke quinone n'ime RC nwere ike ịnwe oke karịa. Iji nwalee ma protein W na-emetụta mgbanwe nke quinones site na RC, anyị mere nnwale oxidation cytochrome na mkpokọta ubiquinone 2 (UQ2) (ihe atụ nke UQ10 eke nwere ọdụ isoprene dị mkpụmkpụ) (Foto 2E). Ọ bụ ezie na ọnụnọ nke chelated quinone na-egbochi mkpebi ziri ezi nke doro anya Michaelis constant (RC-LH114-W na RC-LH116 dabara adaba maka 0.2±0.1μM na 0.5±0.2μM, n'otu n'otu), ọnụego kachasị elu nke RC-LH114-W (4.6±0.2 e-RC-1 s-1) karịrị 28±5% karịa RC-LH116 (3.6±0.1 e-RC-1 s-1).
Anyị buru ụzọ mee atụmatụ na protein-W dị n'ihe dị ka 10% nke isi ihe dị mgbagwoju anya (16); ebe a, ọnụego obibi nke mkpụrụ ndụ na-eto eto dị obere, nke dị ọkara, na nke dị elu bụ 15±0.6%, 11±1% na 0.9±0.5, n'otu n'otu % (Foto 2F). Ntụnyere ọnụọgụ nke mass spectrometry gosiri na mgbakwunye nke histidine tag ebelataghị oke protein-W ma e jiri ya tụnyere ụdị anụ ọhịa (P = 0.59), yabụ ọkwa ndị a abụghị ihe e ji mee protein-W agbanwere agbanwe (Foto S10). Agbanyeghị, obere ọnụnọ protein-W a na RC-LH1 complex nwere ike ikwe ka ụfọdụ RCs tụgharịa na ọsọ ọsọ, si otú a na-ebelata mgbanwe quinone/quinolone dị nwayọ na RC-LH116 complex. Anyị hụrụ na oke nnyefe ìhè dị elu ekwekọghị na data transcriptomics na-adịbeghị anya, nke na-egosi na ngosipụta mkpụrụ ndụ pufW na-abawanye n'okpuru ìhè siri ike (Foto S11) (23). Ihe dị iche n'etiti ntinye akwụkwọ pufW na ntinye protein-W n'ime mgbagwoju anya RC-LH1 na-agbagwoju anya ma nwee ike igosipụta usoro mgbagwoju anya nke protein ahụ.
Na RC-LH114-W, e kewara cardiolipin 6 (CDL), phosphatidylcholine 7 (POPC), 1 phosphatidylglycerol (POPG) na 29 β-DDM molekul n'ime ya ma gosipụta ha CDL 6, POPC 24, POPG 2 na βDDM 12. RC-LH116 (Foto 5, A na B). N'ime ihe owuwu abụọ a, CDL fọrọ nke nta ka ọ dị n'akụkụ cytoplasmic nke ihe mgbagwoju anya ahụ, ebe POPC, POPG na β-DDM dịkarịsịrị n'akụkụ luminal. E kewapụrụ molekul lipid na sater abụọ na mpaghara αβ-1 ruo αβ-6 nke ihe mgbagwoju anya RC-LH114-W (Foto 5A), e kewapụkwara ise na mpaghara nhata nke RC-LH116 (Foto 5B). A hụrụ ọtụtụ lipids n'akụkụ nke ọzọ nke mgbagwoju anya ahụ, ọkachasị CDL, nke a chịkọtara n'etiti RC na αβ-7 ruo αβ-13 (Foto 5, A na B). Lipids na ihe nhicha ndị ọzọ edoziri edozi dị n'èzí mgbanaka LH1, agbụ acyl ndị edoziri nke ọma na-agbatị n'etiti obere LH1, nke a na-akpọ β-DDM na RC-LH114-W, ma kọwaa dị ka β-DDM na ngwakọta RC A nke β-DDM na POPC-LH116. Ọnọdụ ndị yiri ya nke chelating lipids na ihe nhicha n'ime nhazi anyị na-egosi na ha bụ ebe njikọ physiological (Foto S12A). Ọnọdụ nke molekul ndị yiri ya na Tch nwekwara ezigbo nguzozi. Dị nro na Trv. Mkpụrụ ndụ 970 RC-LH1 (Foto S12, B ruo E) (9, 12) na ihe fọdụrụ n'ime haịdrojiin nke otu isi lipid gosiri ezigbo nchekwa na nhazi usoro (Foto S13), na-egosi na CDL echekwara nke na-ejikọ na RC (24), ebe ndị a nwere ike ịchekwa na mgbagwoju RC-LH1.
(A na B) E ji katọn na-egosi peptides RC-LH114-W (A) na RC-LH116 (B), a na-egosikwa pigments site na mkpara, site na iji usoro agba dị na Foto 1. A na-egosi lipids na uhie, a na-egosikwa ihe nhicha na isi awọ. UQ nke ejikọtara na saịtị RC QA na QB bụ odo odo, ebe UQ nke dịpụrụ adịpụ bụ acha anụnụ anụnụ. (C na D) Otu echiche dị ka (A) na (B), ebe ewepụrụ lipids. (E ruo G) Echiche buru ibu nke Q1(E), Q2(F) na Q3(G) site na RC-LH116, yana agbụ akụkụ nke na-emetụta ibe ha. A na-egosi njikọ hydrogen dị ka ahịrị ojii nwere akara.
Na RC-LH116, ma RC QA na QB UQ, ndị na-esonye na nnyefe eletrọn na usoro nkewa chajị, na-agbaze n'ebe njikọ ha. Agbanyeghị, na RC-LH114-W, edozibeghị quinone QB ma a ga-atụle ya nke ọma n'okpuru. Na mgbakwunye na QA na QB quinones, a na-ekenye molekul UQ abụọ (dị n'etiti mgbaaka RC na LH1) na nhazi RC-LH114-W dịka otu isi ha siri dị (dị na Q1 na Q2, n'otu n'otu). Foto 5C). E kenyere nkeji isoprene abụọ na Q1, maapụ njupụta ahụ na-edozi ọdụ isoprene iri zuru oke nke Q2. Na nhazi nke RC-LH116, edoziri molekul UQ10 atọ (Q1 ruo Q3, Foto 5D) na molekul niile nwere njupụta doro anya n'akụkụ ọdụ ahụ (Foto 5, D ruo G). N'ime usoro abụọ ahụ, ọnọdụ nke otu isi quinone nke Q1 na Q2 nwere nguzozi dị mma (Foto S12F), ha na-akpakọrịta naanị na RC. Q1 dị n'ọnụ ụzọ nke oghere W nke RC-LH114-W (Foto 1G na 5, C, D na E), Q2 dịkwa nso na ebe njikọ QB (Foto 5, C, D) na F). Ihe fọdụrụ L-Trp143 na L-Trp269 dị nso na Q1 na Q2 ma na-enye mmekọrịta π nwere ike itinye (Foto 5, E na F, na Foto S12). L-Gln88, 3.0 Å site na oxygen dị anya nke Q1, na-enye njikọ hydrogen siri ike (Foto 5E); ihe fọdụrụ a na-echekwa na RC niile ma e wezụga mmekọrịta kachasị anya (Foto S13). A na-eji nlezianya dochie L-Ser91 maka Thr n'ọtụtụ RC ndị ọzọ (Foto S13), bụ 3.8 Angstroms site na methyl oxygen nke Q1, ọ nwekwara ike inye njikọ hydrogen na-adịghị ike (Foto 5E). Q3 adịghị ka ọ nwere mmekọrịta kpọmkwem, mana ọ dị na mpaghara hydrophobic n'etiti subunit RC-M na subunit LH1-α 5 ruo 6 (Foto 5, D na G). A doziela Q1, Q2 na Q3 ma ọ bụ chelated quinones dị nso na Tch. Gentle, Trv. Strain 970 na Blc. Ọdịdị iris (9, 10, 12) na-atụ aka na ebe njikọ quinone enyemaka echekwara na RC-LH1 complex (Foto S12G). UQ ise gbawara agbawa na RC-LH116 kwekọrọ na 5.8±0.7 nke mgbagwoju ọ bụla nke ejiri nnukwu arụmọrụ mmiri mmiri (HPLC) chọpụta, ebe UQ atọ gbawara agbawa na RC-LH114-W dị ala karịa Uru a tụrụ nke 6.2±0.3 (Fig. S14) na-egosi na enwere molekul UQ a ​​na-edozighị edozi na nhazi ahụ.
Polypeptide L na M nke ọ bụla nwere TMH ise ma mepụta heterodimer nke na-ejikọta otu BChl dimer, monomer BChl abụọ, monomer bacteriophage abụọ (BPh), na otu iron na-abụghị Heme na otu ma ọ bụ abụọ UQ10 molekul. Site na ọnụnọ nke njikọ hydrogen na otu ketone ikpeazụ na mkpokọta ya a maara na Rps, a na-etinye carotenoids na M-subunit, nke a na-akpọ cis-3,4-dehydroorhodopin. Ụdị (25). A na-ejikọta mpaghara mpụta nke RC-H na membrane site na otu TMH. Nhazi RC zuru oke yiri RC subunit atọ nke ụdị ndị metụtara ya (dịka Rba). sphaeroides (PDB ID: 3I4D). Macrocycles nke BChl na BPh, azụ carotenoid na ígwè na-abụghị heme na-adakọ n'ime oke mkpebi nke usoro ndị a, dịka otu isi UQ10 na saịtị QA na quinone QB na RC-LH116 (Foto S15).
Ọdịdị nke ihe owuwu RC abụọ nwere ọnụego ọnụnọ QB dị iche iche na-enye ohere ọhụrụ iji nyochaa mgbanwe nhazi na-aga n'ihu na njikọ QB quinone. Na mgbagwoju RC-LH116, quinone QB dị n'ọnọdụ "nso" zuru oke (26), mana nkewa nke RC-LH114-W enweghị quinone QB. Enweghị quinone QB na RC-LH114-W, nke bụ ihe ijuanya n'ihi na mgbagwoju anya ahụ na-arụ ọrụ, karịa mgbagwoju RC-LH116 nwere quinone QB edoziri nke ọma. Ọ bụ ezie na mgbaaka LH1 abụọ ahụ na-eme ihe dị ka quinone isii, a na-edozi ise n'usoro na mgbanaka RC-LH116 mechiri emechi, ebe naanị atọ ka a na-ejedebe na nhazi na mgbanaka RC-LH114-W mepere emepe. Nsogbu nhazi a mụbara nwere ike igosipụta nnọchi ngwa ngwa nke saịtị RC-LH114-W QB, kinetics quinone ngwa ngwa na mgbagwoju anya ahụ, na ohere ịbawanye nke ịgafe aka LH1. Anyị na-atụ aro na enweghị UQ na saịtị RC QB nke RC-LH114-W nwere ike ịbụ nsonaazụ nke mgbagwoju anya na nke na-arụ ọrụ karịa, ebe QB nke RC-LH114-W akpọnwụwo ozugbo na ntụgharị UQ. Ogbo a kapịrị ọnụ (emechiela ọnụ ụzọ ámá nke saịtị QB) na-egosipụta nhazi nke ọrụ a.
Na-enweghị QB, ntụgharị L-Phe217 na-eso ya gaa n'ọnọdụ na-ekwekọghị na njikọ UQ10, n'ihi na ọ ga-akpata nkpọkọ oghere na nkeji isoprene mbụ nke ọdụ (Foto 6A). Na mgbakwunye, mgbanwe nhazi bụ isi doro anya doro anya, ọkachasị helix de (helix dị mkpụmkpụ na loop dị n'etiti TMH D na E) ebe a na-agbanwe L-Phe217 gaa na akpa njikọ QB na ntụgharị nke L-Tyr223 (Foto 6A) Iji mebie njikọ hydrogen na usoro M-Asp45 ma mechie ọnụ ụzọ nke ebe njikọ QB (Foto 6B). Helix de na-atụgharị na ntọala ya, a na-agbanwe Cα nke L-Ser209 site na 0.33Å, ebe a na-agbanwe L-Val221Cα site na 3.52Å. Enweghị mgbanwe a na-ahụ anya na TMH D na E, nke a na-apụghị izere ezere na nhazi abụọ ahụ (Foto 6A). Dịka anyị maara, nke a bụ nhazi mbụ dị na RC eke nke na-emechi ebe QB. Ntụnyere ya na nhazi zuru oke (nke jikọtara QB) na-egosi na tupu quinone ebelata, a chọrọ mgbanwe nhazi iji mee ka ọ banye na quinone. L-Phe217 na-agbagharị iji mepụta mmekọrịta π na otu isi quinone, helix na-agbanwekwa gaa n'èzí, na-ekwe ka ọkpụkpụ nke L-Gly222 na agbụ akụkụ nke L-Tyr223 mepụta netwọk njikọ hydrogen nwere nhazi njikọ hydrogen kwụsiri ike (Foto 6, A na C).
(A) Katuunu nke hologram (agbụ ígwè L, agbụ ígwè oroma/M, magenta) na usoro apo (isi awọ) na-ekpuchi, ebe a na-egosipụta ihe fọdụrụ dị mkpa n'ụdị ihe nnọchianya dị ka mkpanaka. UQ10 na-anọchi anya site na ogwe odo odo. Ahịrị ntụpọ ahụ na-egosi njikọ hydrogen nke e guzobere na nhazi ahụ dum. (B na C) Nnọchite anya elu nke apolipoprotein na nhazi mgbanaka dum, na-egosipụta oxygen agbụ ígwè nke L-Phe217 na acha anụnụ anụnụ na L-Tyr223 na uhie, n'otu n'otu. Akụkụ L bụ oroma; akụkụ M na H anaghị agba agba. (D na E) Apolipoprotein (D) na saịtị RC QB dum [agba site na (A) n'otu n'otu] na Thermophilus thermophilus PSII (akwụkwọ ndụ akwụkwọ ndụ, acha anụnụ anụnụ nwere quinone plastik; PDB ID: 3WU2) Hazie (58).
N'ụzọ a na-atụghị anya ya, ọ bụ ezie na enwere ọtụtụ nhazi nke RC ndị na-enweghị QB na-enweghị LH1, akọpụtabeghị mgbanwe nhazi nke a hụrụ na ọmụmụ ihe a na mbụ. Ndị a gụnyere nhazi mbelata QB sitere na Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) na Rba. sphaeroides (PDB ID: 1OGV) (29), ihe niile fọrọ nke nta ka ọ bụrụ otu ihe ahụ dị ka nhazi QB ha dum. Nnyocha nke 3PRC nke ọma gosiri na mkpụrụ ndụ ihe nketa LDAO (Lauryl Dimethyl Amine Oxide) na-ejikọ n'ọnụ ụzọ nke ọnọdụ QB, nke nwere ike igbochi nhazigharị ka ọ bụrụ nhazi mechiri emechi. Ọ bụ ezie na LDAO anaghị ere ere n'otu ọnọdụ na 1EYS ma ọ bụ 1OGV, a na-eji otu ncha ahụ kwadebe RC ndị a ma ya mere ha nwere ike imepụta otu mmetụta ahụ. Nhazi kristal nke Rba. Sphaeroides RC nke ejiri cytochrome c2 (PDB ID: 1L9B) mee ka ọ dịkwa ka o nwere ebe QB mechiri emechi. Agbanyeghị, n'ọnọdụ a, mpaghara N-terminal nke polypeptide RC-M (na-akpakọrịta na ebe njikọ QB site na njikọ H nke ihe fọdụrụ Tyr na helix Q) na-anabata nhazi na-adịghị ahụkebe, a kabeghịkwa enyocha mgbanwe nhazi QB ọzọ (30). Ihe na-eme ka obi sie anyị ike bụ na anyị ahụbeghị ụdị mgbanwe a nke polypeptide M na nhazi RC-LH114-W, nke fọrọ nke nta ka ọ bụrụ otu ihe ahụ dị ka mpaghara N-terminal nke RC-LH116 RC. E kwesịkwara ịmara na mgbe ewepụsịrị antenna LH1 nke e ji sachaa ihe, edoziri RCs apolipoprotein dị na PDB, nke wepụrụ ọdọ mmiri quinone dị n'ime na lipids n'ime oghere dị n'etiti RC na elu ime nke mgbanaka LH1 gbara ya gburugburu (31, 32). RC ka na-arụ ọrụ n'ihi na ọ na-ejigide ihe niile dị na ya, ma e wezụga quinone QB nke a na-agbaze agbaze, nke na-adịghị akwụsi ike ma na-efunahụkarị n'oge nkwadebe (33). Na mgbakwunye, a maara na iwepụ LH1 na lipids cyclic eke sitere na RC nwere ike inwe mmetụta na ọrụ, dị ka ogologo oge ndụ nke steeti P+QB nke kewapụrụ chajị (31, 34, 35). Ya mere, anyị na-eche na ịdị adị nke mgbanaka LH1 mpaghara gbara RC gburugburu nwere ike ịnọgide na-enwe ebe QB "emechire emechi", si otú a na-echekwa gburugburu ebe obibi dị nso na QB.
Ọ bụ ezie na apolipoprotein (na-enweghị QB quinone) na nhazi zuru oke na-anọchite anya naanị foto abụọ nke ntụgharị nke saịtị QB, kama usoro ihe omume, enwere ihe ngosi na enwere ike itinye njikọ ahụ iji gbochie njikọta ọzọ site na hydroquinone Iji gbochie mgbochi substrate. Mmekọrịta nke quinolol na quinone dị nso na saịtị QB nke apolipoprotein nwere ike ịdị iche, nke na-eduga na RC ịjụ ya. A na-atụ aro ogologo oge na mgbanwe nhazi na-arụ ọrụ na njikọ na mbelata nke quinones. Ike nke RC oyi kpọnwụrụ akpọnwụ iji belata quinones mgbe mgbanwe gbara ọchịchịrị gasịrị emebiela (36); X-ray kristal na-egosi na mmebi a bụ n'ihi na quinones QB na-ejide na nhazi "distal" ihe dị ka 4.5 Å site na ọnọdụ proximal na-arụ ọrụ (26), 37). Anyị na-atụ aro na nhazi njikọ a bụ foto nke ọnọdụ etiti dị n'etiti apolipoprotein na nhazi mgbanaka zuru oke, nke na-esochi mmekọrịta mbụ na quinone na mmeghe nke saịtị QB.
Ụdị RC II a hụrụ na PSII complex nke ụfọdụ nje bacteria fototrophic na cyanobacteria, algae na osisi nwere nchekwa nhazi na ọrụ (38). Nhazi nhazi egosiri na Foto 6 (D na E) na-emesi ike myirịta dị n'etiti PSII RCs na ebe QB nke mgbagwoju RC nje. Ntụnyere a abụrụla ihe nlereanya ogologo oge maka ịmụ usoro njikọ quinone na mbelata. Akwụkwọ ndị gara aga tụrụ aro na mgbanwe nhazi na-esochi mbelata PSII nke quinones (39, 40). Ya mere, na-atụle nchekwa evolushọn nke RC, usoro njikọ a a na-ahụbeghị mbụ nwekwara ike ịdị irè na ebe QB nke PSII RC na osisi phototrophic oxygenated.
Rps ΔpufW (nhichapụ pufW a na-akpọghị aha) na PufW-His (C-terminal 10x His-tagged protein-W nke a na-egosipụta site na nje pufW locus eke). A kọwara palustris CGA009 n'ọrụ anyị gara aga (16). E nwetara ụdị ndị a na nne na nna ụdị isogenic site na friza site na ịpịa obere ọnụọgụ nke mkpụrụ ndụ na PYE (lita 5 g ọ bụla -1) (echekwara na LB na -80 °C, nwere protein 50% (w/v) glycerol), yist extract na succinate) agar [1.5% (w/v)] efere. E tinyere efere ahụ n'ime akpa n'abalị n'ọchịchịrị na okpomọkụ ụlọ n'okpuru ọnọdụ anaerobic, wee jiri ìhè ọcha (~50 μmolm-2 s-1) nke OSRAM 116-W halogen bulbs (RS Components, UK) nyere maka ụbọchị 3 ruo 5 ruo 5 ruo mgbe otu colony pụtara. E jiri otu ogige ọgwụ gbaa ọgwụ M22+ ọkara (41) nke e tinyere 0.1% (w/v) nke casamino acids (nke a ga-akpọ M22 ugbu a). A kụrụ ọdịbendị ahụ n'okpuru obere ikuku oxygen n'ọchịchịrị na 34°C site na ịma jijiji na 180 rpm ruo awa 48, wee gbanye 70 ml nke ọdịbendị ahụ n'okpuru otu ọnọdụ ahụ ruo awa 24. A na-eji ọdịbendị semi-aerobic nwere olu nke 1 ml gbaa ọgwụ M22 30 ml n'ime karama iko 30 ml zuru ụwa ọnụ nke nwere skru-top ma gbanye ya ọkụ (~50μmolm-2 s-1) ruo awa 48 site na ike magnetik sterile Stirring rod. Mgbe ahụ, a gbara ọgwụ 30 ml nke ọdịbendị ahụ ihe dị ka lita 1 nke ọdịbendị n'okpuru otu ọnọdụ ahụ, nke e jirizie gbaa ọgwụ ihe dị ka lita 9 nke ọdịbendị ọkụ na ~200 μmolm-2 s-1 ruo awa 72. E jiri centrifugation na 7132 RCF wepụta mkpụrụ ndụ ndị ahụ ruo nkeji iri atọ, tinyeghachi ha na ~10 ml nke 20 mM tris-HCl (pH 8.0), ma debe ha na -20°C ruo mgbe ọ dị mkpa.
Mgbe agbazechara, tinye ụfọdụ kristal nke deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) na mbadamba Roche holoenzyme protease inhibitor abụọ (Merck, UK) na mkpụrụ ndụ ndị a kpọchiri akpọchi. N'ime sel nrụgide French psi 20,000 psi (Aminco, USA), a gbawara mkpụrụ ndụ ahụ ugboro 8 ruo 12. Mgbe ewepụrụ mkpụrụ ndụ ndị na-agbawa agbawa na ihe ndị na-agbawa agbawa site na centrifugation na 18,500 RCF maka nkeji iri na ise na 4°C, a kpọpụtara akpụkpọ ahụ site na lysate agba site na centrifugation na 113,000 RCF maka awa abụọ na 43,000°C. Tụfuo akụkụ ahụ na-agbaze agbawa ma tinyeghachi akpụkpọ ahụ agba na 100 ruo 200 ml nke 20 mM tris-HCl (pH 8.0) ma mee ka ọ dị ka otu ruo mgbe enweghị ihe ndị a na-ahụ anya. E tinyere akpụkpọ ahụ a kwụgidere n'ime 20 mM tris-HCl (pH 8.0) (Anatrace, USA) nke nwere 2% (w/v) β-DDM ruo otu awa n'ọchịchịrị na 4°C na-akpali ya nke ọma. Mgbe ahụ, centrifuge na 70°C iji gbazee 150,000 RCF na 4°C maka otu awa iji wepụ ihe ndị na-anaghị agbaze.
E tinyere akpụkpọ ahụ na-agbaze mmiri site na nje ΔpufW na kọlụm mgbanwe ion DEAE Sepharose 50 ml nwere olu kọlụm atọ (CV) nke ihe nchekwa njikọ [20 mM tris-HCl (pH 8.0) nwere 0.03% (w / v) β-DDM]. Jiri ihe nchekwa njikọ CV abụọ saa kọlụm ahụ, wee jiri ihe nchekwa njikọ abụọ nwere 50 mM NaCl saa kọlụm ahụ. E jiri gradient linear nke 150 ruo 300 mM NaCl (na ihe nchekwa njikọ) gbasaa mgbagwoju anya RC-LH116 na 1.75 CV, e jiri ihe nchekwa njikọ nke nwere 300 mM NaCl na 0.5 CV gbasaa mgbagwoju njikọ fọdụrụ. Chịkọta ụdịrị absorption dị n'etiti 250 na 1000 nm, debe akụkụ ahụ nwere oke absorbance (A880/A280) karịa 1 na 880 ruo 280 nm, gwakọta ya ugboro abụọ na ihe nchekwa njikọ, ma jiri otu usoro ahụ ọzọ na kọlụm DEAE On cleansing. Gwakọta akụkụ ndị ahụ na oke A880/A280 karịa 1.7 na A880/A805 karịa 3.0, mee agba nke atọ nke mgbanwe ion, ma debe akụkụ ndị nwere oke A880/A280 karịa 2.2 na A880/A805 karịa 5.0. A tụbara ihe mejupụtara ahụ nke a sachara nke ọma ruo ~2 ml n'ime ihe nzacha centrifugal Amicon 100,000 molekul weight cut-off (MWCO) (Merck, UK), ma tinye ya na kọlụm Superdex 200 16/600 size exclusion (GE Healthcare, US) nke nwere 200 mM NaCl buffer, wee wepụ ya n'otu buffer ahụ na 1.5 CV. Chịkọta spectra absorption nke fraction exclusion size, ma tinye spectra absorption na rate A880/A280 karịa 2.4 na A880/A805 karịa 5.8 ruo 100 A880, ma jiri ha ozugbo maka nkwadebe ma ọ bụ nchekwa grid cryo-TEM Debe na -80°C ruo mgbe ọ dị mkpa.
A na-etinye akpụkpọ ahụ na-agbaze mmiri site na PufW-His na kọlụm 20 ml HisPrep FF Ni-NTA Sepharose (20 mM tris-HCl (pH 8.0) nke nwere 200 mM NaCl na 0.03% (w/w)) na IMAC buffer (GE Healthcare). v) β-DDM). A na-asa kọlụm ahụ na CV ise nke IMAC buffer, wee jiri CV ise nke IMAC buffer nwere 10 mM histidine. E wepụrụ isi ihe mejupụtara ahụ site na kọlụm ahụ na buffer IMAC ise nwere histidine 100 mM. A na-etinye akụkụ nwere RC-LH114-W complex ahụ ruo ~10 ml na tankị a kpụkọrọ akpụkọ nke nwere nzacha Amicon 100,000 MWCO (Merck, UK), gwakọta ya ugboro 20 na ihe nchekwa njikọ, wee tinye ya na 25 ml Na kọlụm DEAE Sepharose, a na-eji CV anọ ejikọtara na ihe nchekwa ahụ tupu oge eruo. Jiri ihe nchekwa njikọ CV anọ saa kọlụm ahụ, wee wepụ ihe mgbagwoju anya ahụ na CV asatọ na gradient linear nke 0 ruo 100 mM NaCl (na ihe nchekwa njikọ), yana CV anọ ndị ọzọ nwere ihe nchekwa njikọ 100 mM. Ihe mgbakwụnye fọdụrụnụ nke e si na sodium chloride gbakọta ya na oke A880/A280 dị elu karịa 2.4 na oke A880/A805 dị elu karịa akụkụ 4.6 ka e tinyere na ~2 ml na nzacha centrifugal Amicon 100,000 MWCO, ma jupụta na 1.5 CV IMAC Buffer nke e mere ka ọ dị nha Superdex 200 16/600, wee wepụ ya na otu ihe nchekwa ahụ karịa 1.5 CV. Chịkọta spectra absorption nke akụkụ mwepụ nha ma tinye spectra absorption na oke A880/A280 karịa oke 2.1 na A880/A805 karịa oke 4.6 ruo 100 A880, nke a na-eji ozugbo maka nkwadebe grid TEM kpọnwụrụ akpọnwụ ma ọ bụ chekwaa na -80°C ruo mgbe ọ dị mkpa.
E jiri friza Leica EM GP mikpuru ihe dị ka obere okpomọkụ kwadebe grid TEM dị iche iche. A gwakọtara ihe mgbagwoju ahụ na IMAC buffer ruo A880 nke 50, wee tinye 5μl n'elu QUANTIFOIL 1.2/1.3 ọla kọpa a kpụchara akpụcha ọhụrụ (Agar Scientific, UK). Kpoo grid ahụ na 20°C na iru mmiri dị 60% maka sekọnd 30, wee hichaa ya nkụ maka sekọnd 3, wee gbanyụọ ya na mmiri mmiri ethane na -176°C.
E dekọrọ data nke mgbagwoju anya RC-LH114-W na eBIC (Electronic Bioimaging Center) (British Diamond Light Source) site na iji igwe onyonyo Titan Krios, nke na-arụ ọrụ na voltaji na-agba ọsọ nke 300kV, yana mmụba aha nke 130,000 × na ike nke- Họrọ oghere nke 20 eV. E jiri Gatan 968 GIF Quantum nwere ihe nchọpụta elu K2 dekọọ onyonyo na ọnọdụ ịgụta iji chịkọta data. Nha pixel e mere ka ọ dị nha bụ 1.048Å, ọnụego dose ahụ bụkwa 3.83 e-Å-2s-1. E chịkọtara ihe nkiri ahụ n'ime sekọnd 11 wee kewaa ya n'ime akụkụ 40. Jiri mpaghara mkpuchi carbon ahụ mee ka igwe onyonyo ahụ lekwasị anya ọzọ, wee chịkọta fim atọ kwa oghere. Na mkpokọta, anakọtara fim 3130, yana ụkpụrụ defocus dị n'etiti -1 na -3μm.
E ji otu igwe onyonyo ahụ chịkọta data maka usoro RC-LH116 na Asterbury Biostructure Laboratory (Mahadum Leeds, UK). E chịkọtara data ahụ na ụdị agụta ya na mmụba nke 130 k, e wee hazie nha pixel ahụ na 1.065 Å na dose nke 4.6 e-Å-2s-1. E dekọrọ ihe nkiri ahụ n'ime sekọnd 12 ma kewaa ya n'ime akụkụ 48. Na mkpokọta, e chịkọtara ihe nkiri 3359, yana uru defocus dị n'etiti -1 na -3μm.
A na-eme nhazi data niile na pipeline Relion 3.0 (42). Jiri Motioncorr 2 (43) dozie mmegharị ahụ site na ịtụ nha dose, wee jiri CTFIND 4.1 (44) chọpụta paramita CTF (ọrụ mbufe ọdịiche). E gosiri fotomikrographs nkịtị mgbe usoro nhazi mbụ ndị a gasịrị na Foto 2. S16. A na-emepụta template nhọrọ akpaka site na iji aka họrọ ihe dị ka pikselụ 250 nke mkpụrụ 1000 na etiti pikselụ 250 na enweghị nhazi akụkụ abụọ (2D), si otú a na-ajụ nhazi ndị ahụ na-ezute mmetọ sample ma ọ bụ enweghị njirimara a na-ahụ anya. Mgbe ahụ, emere nhọrọ akpaka na microphotographs niile, RC-LH114-W bụ mkpụrụ 849,359, mgbagwoju RC-LH116 bụ mkpụrụ 476,547. A na-emerị ihe niile ahọpụtara agba abụọ nke nhazi 2D na-abụghị ntụaka, mgbe nke ọ bụla gasịrị, a na-ajụ ihe ndị dabara na mpaghara carbon, mmetọ ihe nlele, enweghị ihe doro anya ma ọ bụ ihe ndị na-ekpuchi nke ọma, nke na-eme ka 772,033 (90.9%) na 359,678 (75.5%) A na-eji ihe ndị ahụ eme ihe maka nhazi 3D nke RC-LH114-W na RC-LH116 n'otu n'otu. E mepụtara ihe nlereanya ntụaka 3D mbụ site na iji usoro stochastic gradient down. Site na iji ihe nlereanya mbụ dị ka ntụaka, a na-ekewa ihe ndị ahụ ahọpụtara n'ime ụdị anọ na 3D. Site na iji ihe nlereanya dị na ụdị a dị ka ntụaka, mee nhazi 3D na ihe ndị ahụ dị na ụdị kachasị ukwuu, wee jiri ihe nzacha 15Å mbụ dị ala iji kpuchie mpaghara ihe mgbaze, tinye pikselụ 6 nke nsọtụ dị nro, ma tinyezie pikselụ ahụ iji dozie ọrụ mbufe Modulation kacha elu nke ihe nchọpụta elu. Maka dataset RC-LH114-W, emezigharịrị ihe nlereanya mbụ a site na iwepụ njupụta siri ike dị n'akụkụ ihe mkpuchi ihu (anaghị ejikọta ya na njupụta mgbagwoju anya dị na UCSF Chimera). A na-eji ihe nlereanya ndị a (mkpebi nke RC-LH114-W na RC-LH116 bụ 3.91 na 4.16 Å, n'otu n'otu) dị ka ntụaka maka agba nke abụọ nke nhazi 3D. A na-ejikọta ihe ndị ejiri mee ihe na klas 3D mbụ ma enweghị njikọ siri ike na agbataobi ahụ. Nchikota ma ọ bụ enweghị atụmatụ nhazi doro anya. Mgbe agba nke abụọ nke nhazi 3D gasịrị, ahọpụtara ụdị nwere mkpebi kachasị elu [Maka RC-LH114-W, otu ụdị bụ 377,703 irighiri (44.5%), maka RC-LH116, enwere ụdị abụọ, ngụkọta nke 260,752 irighiri (54.7%) , Ebe ha bụ otu naanị mgbe ejikọtara ha mgbe ntụgharị mbụ gasịrị na obere ọdịiche]. A na-ewepụta ihe ndị ahọpụtara ọzọ n'ime igbe pikselụ 400 ma jiri nhazi 3D mee ka ha dị mma. A na-eji nzacha 15Å mbụ dị ala, mgbasawanye maapụ pikselụ 3 na ihe mkpuchi dị nro pikselụ 3 emepụta ihe mkpuchi ihe mgbaze ahụ. Site na iji mmezi CTF kwa-particle, mmezi mmegharị kwa-particle na agba nke abụọ nke mmezi CTF kwa-particle, mmezi 3D, ihe mkpuchi ihe mgbaze na nhazi mgbe emechara nzọụkwụ ọ bụla iji mee ka udidi ahụ pụtakwuo mma. Site na iji uru njedebe FSC (Fourier Shell Correlation Coefficient) nke 0.143, mkpebi nke ụdị ikpeazụ nke RC-LH114-W na RC-LH116 bụ 2.65 na 2.80Å, n'otu n'otu. E gosiri usoro FSC nke ụdị ikpeazụ na Foto 2. S17.
E si na UniProtKB budata usoro protein niile: 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). E jiri SWISS-MODEL (45) wuo ụdị nhazi nke RC, nke nwere usoro protein nke RC-L, RC-M na RC-H na nhazi kristal nke Rba. Ejiri sphaeroides RC mee ihe dị ka template (PDB ID: 5LSE) (46). Jiri ngwa "map dabara adaba" dị na UCSF Chimera iji mee ka ihe nlereanya emepụtara dabara na maapụ (47), melite nhazi protein, na cofactor [4×BChl a (aha residue library monomer = BCL), 2×BPh a (BPH), otu ma ọ bụ ụdị UQ10 abụọ (U10), otu iron na-abụghị heme (Fe) na otu 3,4-dihydrohexacarbonylcholine (QAK)] jiri Coot (48) tinye. Ebe ọ bụ na QAK adịghị na ọbá akwụkwọ monomer, e jiri ngwa eLBOW dị na PHENIX (49) mee ka ọ dị mfe.
Mgbe nke ahụ gasịrị, e wuru obere LH1. Na mbụ, e jiri ngwa ọrụ owuwu akpaka na PHENIX (49) wuo akụkụ nke usoro LH1 na akpaghị aka site na iji map na usoro protein LH1-α na LH1-β dị ka ntinye. Họrọ obere LH1 zuru oke, wepụta ya ma tinye ya na Coot, tinye usoro na-efu na ya aka, ma jiri aka hazie nhazi ahụ dum tupu ịtinye BCls a (BCL) abụọ na spirilloxanthin (CRT) [dịka njupụta Rps nke mgbagwoju anya LH1 dị mkpa na ọdịnaya carotenoid a maara si dị. Ụdị (17)]. Detuo obere LH1 zuru oke, ma jiri UCSF Chimera "Ngwaọrụ Maapụ Docking" dochie mpaghara na-abụghị ụdị nke njupụta LH1 dị nso, wee mee ka ọ dị mma na Coot; megharịa usoro ahụ ruo mgbe e mere ụdị obere LH1 niile. Maka nhazi RC-LH114-W, site na iwepụta njupụta a na-ekeghị na Coot, a na-ekewa protein ahụ site na ihe ndị ọzọ na-abụghị protein na map USCF Chimera ma jiri ngwa Autobuild guzobe ụdị mbụ, yana ihe ndị fọdụrụ (protein-W) Modeling. Na PHENIX (49). Tinye usoro ọ bụla na-efu na ụdị ihe si na ya pụta na Coot (48), wee jiri aka hazie subunit dum. Njupụta a na-ekeghị na-adabara njikọta nke lipids (PDB monomer library ID nke CDL = CDL, POPC = 6PL na POPG = PGT), ihe nhicha β-DDM (LMT) na molekul UQ10 (U10). Jiri optimization PHENIX (49) na optimization aka na Coot (48) iji mee ka ụdị mbụ zuru oke ruo mgbe enweghị ike imeziwanye ọnụ ọgụgụ ụdị na ịdị mma anya nke dabara. N'ikpeazụ, jiri LocScale (50) mee ka map mpaghara dị nkọ, wee mee ọtụtụ usoro ndị ọzọ nke ịme ihe nlereanya njupụta a na-ekeghị na optimization akpaka na aka.
E gosiri peptides, cofactors na lipids na quinones ndị ọzọ dị n'ime njupụta ha na Foto 1 na 2. S18 ruo S23. E gosiri ozi ọnụọgụgụ nke ihe nlereanya ikpeazụ na Tebụl S1.
Ọ gwụla ma akọwapụtara ya n'ụzọ ọzọ, a chịkọtara spectra nnabata UV/Vis/NIR na Cary60 spectrophotometer (Agilent, USA) na nkeji 1 nm site na 250 nm ruo 1000 nm na oge njikọta nke 0.1s.
Gwakọta ihe nlele ahụ na quartz cuvette nwere ụzọ 2 mm gaa na A880 nke 1, wee chịkọta ụdị absorption dị n'etiti 400 na 1000 nm. E chịkọtara ụdị dichroic okirikiri ahụ na spectropolarimeter Jasco 810 (Jasco, Japan) na 1 nm n'etiti 400 nm na 950 nm na 20 nm min-1.
A na-ekpebi ọnụọgụgụ mkpochapu molar site na ịgbasa mgbagwoju anya isi na A880 nke ihe dị ka 50. Gbasaa olu 10μl na ihe nchekwa 990μl ma ọ bụ methanol, wee chịkọta ụdị mkpochapu ozugbo iji belata mbibi BChl. A gbakọrọ ọdịnaya BChl nke ihe nlele methanol ọ bụla site na ọnụọgụgụ mkpochapu na 771 nm nke 54.8 mM-1 cm-1, a chọpụtakwara ọnụọgụgụ mkpochapu (51). Kewaa mkpokọta BChl a tụrụ site na 32 (RC-LH114-W) ma ọ bụ 36 (RC-LH116) iji chọpụta mkpokọta mgbagwoju anya isi, nke a na-eji eme ihe iji chọpụta ụdị mkpochapu nke otu ihe nlele anakọtara na ihe nchekwa Extinction coefficient. parallel. E were nha ugboro atọ maka ihe nlele ọ bụla, ejiri nkezi nnabata nke oke BChl Qy mee ihe maka ngụkọ. Ọnụọgụ mbibi nke RC-LH114-W nke a tụrụ na 878 nm bụ 3280±140 mM-1 cm-1, ebe ọnụọgụ mbibi nke RC-LH116 nke a tụrụ na 880 nm bụ 3800±30 mM-1 cm-1.
A tụrụ UQ10 dịka usoro dị na (52) si dị. Na nkenke, ejiri usoro Agilent 1200 HPLC mee HPLC nke na-agbanwe agbanwe (RP-HPLC). Gbazee ihe dị ka 0.02 nmol nke RC-LH116 ma ọ bụ RC-LH114-W na 50μl nke 50:50 methanol:chloroform nwere 0.02% (w/v) ferric chloride, ma tinye Beckman Coulter Ultrasphere ODS 4.6 mm nke e mere ka ọ dị mma. Gbazee na 1 ml-1 min-1 na 40°C na HPLC solvent (80:20 methanol:2-propanol) na kọlụm ×25 cm. Mee isocratic exlution na HPLC solvent iji nyochaa absorbance na 275 nm (UQ10), 450 nm (carotenoids) na 780 nm (BChl) ruo otu awa. E tinyere oke dị na chromatogram 275 nm na nkeji 25.5, nke na-enweghị ihe ndị ọzọ a na-achọpụta. A na-eji mpaghara agbakọtara agbakọọ oke molar nke UQ10 ewepụtara na ntụnyere na usoro nhazi agbakọrọ site na ntinye nke ọkọlọtọ dị ọcha site na 0 ruo 5.8 nmol (Foto S14). A tụlere ihe nlele ọ bụla n'ime ihe atụ atọ, njehie akọwara kwekọrọ na SD nke nkezi.
E ji 30 μM belatara cytochrome obi ịnyịnya (Merck, UK) na 0 ruo 50 μMUQ2 (Merck, UK) kwadebe ihe ngwọta nwere RC-LH1 complex nke nwere oke Qy absorption nke 0.1. E tinyere 30 μM belatara obi ịnyịnya (horse heart cytochrome c2) na 0 ruo 50 μMUQ2 (Merck, UK). E kwadebere ihe nlele 1-ml atọ na mkpokọta UQ2 ọ bụla ma tinye ya n'ime ọchịchịrị n'abalị na 4°C iji hụ na enwere ike ịgbanwe ya kpamkpam na ọchịchịrị tupu a tụọ ya. E tinyere ihe ngwọta ahụ n'ime OLIS RSM1000 modular spectrophotometer nke nwere akara ọkụ/500 nm, ntinye 1.24 mm, oghere etiti 0.12 mm na oghere 0.6 mm. E tinyere ihe nzacha 600 nm ogologo nm n'ọnụ ụzọ fototube ihe nlele na tube fotomultiplier ntụaka iji wepụ ìhè mkpali. E lere anya na nnabata ahụ na 550 nm yana oge njikọta nke 0.15 s. A na-ewepụta ọkụ mkpali site na 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) site na eriri eriri optic na ike 90% site na njikwa DC2200 (Thorlabs Ltd., UK) ma na-ewepụta ya na isi iyi ọkụ na nkuku 90° nke. Ogwe nha ahụ na-emegide enyo iji weghachite ìhè ọ bụla nke ihe nlele ahụ na-abanyeghị na mbụ. Lelee nnabata ahụ 10 s tupu ìhè nke 50 s. Mgbe ahụ, a na-enyocha nnabata ahụ maka 60 s n'ọchịchịrị iji chọpụta oke quinolol na-ebelata cytochrome c23 + n'onwe ya (lee Foto S8 maka data raw).
A haziri data ahụ site n'itinye ọnụego mbụ dị larịị n'ime 0.5 ruo 10 s (dabere na mkpokọta UQ2) na nkezi ọnụego nke ihe nlele atọ niile na mkpokọta UQ2 ọ bụla. Ejiri mkpokọta RC-LH1 nke agbakọrọ site na ọnụọgụ mkpochapụ dị iche iche gbanwee ọnụego ahụ ka ọ bụrụ arụmọrụ catalytic, nke e sere na Origin Pro 2019 (OriginLab, USA), ma tinye ya na ụdị Michaelis-Menten iji chọpụta ụkpụrụ Km na Kcat doro anya.
Maka nha nnabata nwa oge, a gwakọtara ihe nlele RC-LH1 ruo ~2μM na ihe nchekwa IMAC nke nwere sodium ascorbate 50 mM (Merck, USA) na 0.4 mM Terbutin (Merck, USA). A na-eji Ascorbic acid eme ihe dị ka onye na-enye electron àjà, a na-ejikwa tert-butaclofen dị ka ihe mgbochi QB iji hụ na onye na-enye RC bụ isi ka na-ebelata (ya bụ, ọ bụghị photooxidized) n'oge usoro nha. A na-agbakwunye ihe dị ka 3 ml nke ihe nlele na sel na-agbagharị agbagharị (ihe dị ka 0.1 m na dayameta, 350 RPM) yana ogologo ụzọ anya 2 mm iji hụ na ihe nlele dị na ụzọ laser nwere oge zuru oke maka mgbanwe gbara ọchịchịrị n'etiti mkpali mkpali. Jiri ~100-fs laser pulses iji mee ka sistemụ laser Ti: Sapphire dịkwuo elu (Spectra Physics, USA) iji kpalite ihe nlele ahụ na 880 nm na ọnụego ugboro ugboro nke 1 kHz (20 nJ maka NIR ma ọ bụ 100 nJ maka Vis). Tupu ị chịkọta data, kpughee ihe nlele ahụ na ọkụ mkpali ruo ihe dị ka nkeji iri atọ. Ikpughe ga-eme ka QA ghara ịrụ ọrụ (ikekwe belata QA otu ugboro ma ọ bụ ugboro abụọ). Mana biko mara na usoro a nwere ike ịgbanwe n'ihi na mgbe ogologo oge nke mgbanwe ọchịchịrị gasịrị, RC ga-alaghachi nwayọ nwayọ na ọrụ QA. Ejiri Helios spectrometer (Ultrafast Systems, USA) tụọ spectra nwa oge na oge igbu oge nke -10 ruo 7000 ps. Jiri ngwanrọ Surface Xplorer (Ultrafast Systems, USA) wepụ ihe ndị dị na data ahụ, wee jikọta ma hazie ha. Jiri ngwugwu ngwanrọ CarpetView (Light Conversion Ltd., Lithuania) iji setịpụ data ejikọtara iji nweta spectra dị iche iche metụtara ire ere, ma ọ bụ jiri ọrụ nke gụnyere ọtụtụ exponents na nzaghachi ngwaọrụ iji dabara na mgbanwe spectral otu-wavelength na Origin (OriginLab, USA).
Dịka ekwuru n'elu (53), e mere ihe nkiri fotosynthetic nke nwere mgbagwoju anya LH1 nke na-enweghị ma eriri RC na nke dị n'akụkụ LH2. A gwakọtara akpụkpọ ahụ na tris 20 mM (pH 8.0) wee tinye ya n'ime quartz cuvette nwere ụzọ anya 2 mm. E jiri pulse laser 30nJ mee ka ihe nlele ahụ dị ọkụ na 540 nm na oge igbu oge nke -10 ruo 7000 ps. Hazie nhazi data dịka akọwara maka ihe nlele Rps. pal.
A na-eji centrifugation na 150,000 RCF mee ka akpụkpọ ahụ dị nro ruo awa abụọ na 4°C, wee jiri 20 mM tris-HCl na 20 mM NaCl na 200 mM NaCl mee ka akpụkpọ ahụ dị nro. Gbazee akpụkpọ ahụ site n'ịgwakọta ya nwayọ nwayọ na 2% (w/v) β-DDM maka awa 1 n'ọchịchịrị na 4°C. A gwakọtara ihe nlele ahụ na 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) ruo na protein nke 2.5 mg ml-1 (Bio-Rad analysis). Emere nhazi ọzọ site na usoro ebipụtara na mbụ (54), na-amalite site na ịgwakọta protein 50 μg n'ime mkpokọta 50 μl TEAB nwere 1% (w/v) sodium laurate (Merck, UK). Mgbe sonication gasịrị ruo sekọnd 60, e belatara ya na 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) na 37°C maka nkeji 30. Maka S-alkylation, tinye ihe nlele ahụ na 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) ma tinye ya na mmiri isopropanol stock 200 mM maka nkeji 10 na okpomọkụ ụlọ. Emere mgbaze proteolytic site na ịgbakwunye 2 μg trypsin/endoproteinase Lys-C ngwakọta (Promega UK) ma tinye ya na 37°C ruo awa 3. E wepụtara laurate surfactant site na ịgbakwunye 50 μl ethyl acetate na 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) na vortexing maka sekọnd 60. E jiri centrifugation kwalite nkewa nke usoro ahụ na 15,700 RCF maka nkeji ise. Dịka usoro onye nrụpụta si dị, e jiri kọlụm ntụgharị C18 (Thermo Fisher Scientific, UK) mee ka mmiri gbapụta ma wepụ nnu nke ọma n'oge dị ala nke nwere peptide ahụ. Mgbe a kpọchara ya site na centrifugation vacuum, a gbazere ihe nlele ahụ na 0.5% TFA na 3% acetonitrile, a nyochakwara 500 ng site na iji nanoflow RP chromatography yana mass spectrometry site na iji paramita sistemụ akọwapụtara na mbụ.
Jiri MaxQuant v.1.5.3.30 (56) maka njirimara protein na nhazi iji chọọ nchekwa data Rps. palustris proteome (www.uniprot.org/proteomes/UP000001426). E tinyela data proteomics mass spectrometry na ProteomeXchange Alliance site na ebe nchekwa PRIDE partner (http://proteomecentral.proteomexchange.org) n'okpuru njirimara data PXD020402.
Maka nyocha site na RPLC yana electrospray ionization mass spectrometry, e ji ụdị Rps dị iche iche mepụta RC-LH1 complex. Site na iji usoro ebipụtara na mbụ (16), mkpokọta protein emepụtara na mkpụrụ ndụ palustris bụ 2 mg ml-1 na 20 mM Hepes (pH 7.8), 100 mM NaCl na 0.03% (w/v) β- (Bio-Rad analysis)) DDM. Dịka usoro nke onye nrụpụta si dị, jiri ngwa nhicha 2D (GE Healthcare, USA) wepụta 10 μg protein site na usoro mmiri ozuzo, wee gbazee ihe mkpuchi ahụ na 20 μl 60% (v/v) formic acid (FA), 20% (v/v) Acetonitrile na 20% (v/v) mmiri. E jiri RPLC (Dionex RSLC) nyochaa microlita ise yana mass spectrometry (Maxis UHR-TOF, Bruker). Jiri kọlụm MabPac 1.2×100 mm (Thermo Fisher Scientific, UK) maka nkewa na 60°C na 100μlmin -1, yana gradient nke 85% (v / v) solvent A [0.1% (v / v) FA na 0.02% (V/v) TFA mmiri mmiri] gaa na 85% (v/v) solvent B [0.1%(v/v) FA na 0.02%(v/v) na 90%(v/v) acetonitrile TFA] Site na iji isi iyi ionization electrospray ọkọlọtọ na paramita ndabara maka ihe karịrị nkeji 60, spectrometer oke na-enweta 100 ruo 2750 m/z (oke-na-charge). Site n'enyemaka nke ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), maapụ oke spectrum na obere nke mgbagwoju anya ahụ.
A zụlitere mkpụrụ ndụ ndị a ruo awa 72 n'okpuru 100 ml NF-low (10μMm-2 s-1), ọkara (30μMm-2 s-1) ma ọ bụ elu (300μMm-2 s-1). M22 ọkara (M22 ọkara ebe a na-ewepụ ammonium sulfate na sodium acetate dochie sodium succinate) n'ime karama skru-top 100 ml (23). N'ime usoro 30-s ise, a kpụrụ beads iko 0.1 micron na oke olu nke 1:1 iji tee mkpụrụ ndụ ahụ ma jụọ oyi na ice maka nkeji ise. E wepụrụ ihe na-agbazeghị agbaze, mkpụrụ ndụ na-agbazeghị agbaze na beads iko site na centrifugation na 16,000 RCF maka nkeji iri na benchtop microcentrifuge. E kewara akpụkpọ ahụ n'ime rotor Ti 70.1 ya na 100,000 RCF na 20 mM tris-HCl (pH 8.0) yana gradient sucrose 40/15% (w/w) ruo awa iri.
Dịka akọwara n'ọrụ anyị gara aga, nchọpụta ihe mgbochi ọrịa nke akara His na PufW (16). Na nkenke, ihe mejupụtara isi dị ọcha (11.8 nM) ma ọ bụ akpụkpọ ahụ nwere otu njupụta nke RC (nke a na-ekpebi site na oxidation na-ewepụ spectrum dị iche iche ma dakọọ ibu dị na gel agbaze) na 2x SDS loading buffer (Merck, UK) gwakọtara ugboro abụọ. E kewara protein ndị ahụ na gel bis-tris NuPage nke 12% (Thermo Fisher Scientific, UK). E ji Coomassie Brilliant Blue (Bio-Rad, UK) tee gel iji bufee ma hụ akụkụ RC-L. E bufere protein dị na gel nke abụọ na membrane polyvinylidene fluoride (PVDF) nke methanol na-arụ ọrụ (Thermo Fisher Scientific, UK) maka nyocha immunoassay. E mechiri akpụkpọ ahụ PVDF na ntụ mmiri ara ehi 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 na 5% (w / v) nke a kpụchara akpụcha, wee tinye ya na antibody mbụ anti-His (na Dilute the antibody buffer [50 mM tris-HCl (pH 7.6), 150 mM NaCl na 0.05% (v/v) Tween-20] na 1:1000 A190-114A, Bethel Laboratories, USA) ruo awa anọ. Mgbe e sachara ya ugboro atọ ruo nkeji ise na ihe nchekwa antibody, a na-ejikọta akpụkpọ ahụ ya na horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (agbazere 1:10,000 na ihe nchekwa antibody) iji mee ka achọpụta ya (nkeji ise mgbe e sachara ya ugboro atọ na ihe nchekwa antibody) site na iji WESTAR ETA C 2.0 chemiluminescence substrate (Cyanagen, Italy) na Amersham Imager 600 (GE Healthcare, UK).
Site n'ịse ike nke gel ọ bụla e sere agba ma ọ bụ immunoassay lane, ijikọta mpaghara dị n'okpuru oke ahụ ma gbakọọ oke ike nke RC-L (gel agba) na Protein-W (immunoassay), na ImageJ (57) Hazie onyonyo ahụ. A gbanwere oke ndị a ka ọ bụrụ oke molar site n'iche na oke nke RC-L na protein-W na ihe nlele RC-LH114-W dị ọcha bụ 1:1 ma mee ka ntọala data niile dị mma dịka o kwesịrị.
Maka ihe ndị ọzọ gbasara akụkọ a, biko lee 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 Kirmaier, Andrew Hitchcock, C. Neil Hunter
Nhazi dị elu nke mgbagwoju anya nke ọnyà ọkụ 1 dị na ebe mmeghachi omume na-enye nghọta ọhụrụ banyere usoro quinone.
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 Kirmaier, Andrew Hitchcock, C. Neil Hunter
Nhazi dị elu nke mgbagwoju anya nke ọnyà ọkụ 1 dị na ebe mmeghachi omume na-enye nghọta ọhụrụ banyere usoro quinone.
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