Ināianei†Wāhitau o nāianei: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Ko te matatini hauhake mārama o te pokapū tauhohenga 1 (RC-LH1) te wāhanga matua o te whakaahua o ngā huakita whakaahua papura. I whakaurua e mātou ētahi hanganga karuārai-irahiko e rua o te matatini RC-LH1 mai i Rhodopseudomonas palustris. Ko te hanganga taumira 2.65-Å o te matatini RC-LH114-W he 14 ngā koropiko wāhanga LH1 e karapoti ana i te RC, e haukotia ana e te pūmua W, ko te matatini kāore he pūmua-W he hanganga RC katoa e karapotia ana e te RC. He koropiko wāhanga 16 kua kati te LH1. Mā te whakataurite i ēnei hanganga ka kitea ngā āhuatanga o te quinone i roto i te matatini RC-LH1, tae atu ki ngā huringa āhua kāore anō kia whakatauhia i mua i te wā e herea ana te quinone ki te pae RC QB, me te wāhi o ngā pae here quinone āwhina, e āwhina ana ki te tuku i a rātou ki te RC. Ko te hanganga ahurei o te pūmua W ka ārai i te katinga o te koropiko LH1, ā, ka hangaia he awa hei whakatere i te whakawhiti quinone/quinolone.
Ka taea e te pūngao e whakaratohia ana e te photosynthesis te tautoko i te nuinga o ngā mea ora i runga i te whenua, ā, he nui te kaha mō te hangarau koiora o te rā. Ahakoa te whakatairanga i te photosynthesis o te ao, ka whakaatuhia hoki e ngā huakita phototrophic papura ngā momo pūngao me ngā āheinga pākia. Ka taea e rātou te karo i te photosynthesis me te tipu hei huakita heterotrophic i roto i te pōuri, ka taea te whakatika i te hauota me te hauhā waro, te whakaputa hauwai, me te whakaiti i ngā matū kakara (1-3). Hei whakarato pūngao mō ēnei tukanga, me huri tere me te whai hua te mārama ki te pūngao matū. Ka tīmata tēnei tukanga ina mimiti te matatini pūihi hopu-mārama i te mārama me te whakawhiti i te pūngao kua mau ki te pokapū tauhohenga (RC), ka tīmata te wehenga utu (4 – 7). Ko te waeine taketake o te photosynthesis i roto i ngā huakita phototrophic papura he mea tito mai i te momo 2 RC, e karapotia ana e te matatini hauhake-mārama 1 (LH1), e hanga ana i te matatini matua RC-LH1. Ka hangaia te LH1 e te huinga o ngā heterodimers αβ piko, ia o ēnei e herea ana e rua ngā ngota chlorophyll huakita (BChl) a me te kotahi, e rua rānei ngā carotenoids (8-12). Ko te pūihi LH1 māmā rawa he 16, 17 rānei ngā αβ heterodimers e karapoti ana i te RC (9-13) i roto i tētahi porowhita kati, engari i roto i ētahi atu matatini matua, ka whakakorea e ngā peptide transmembrane te haere tonu o te LH1 e karapoti ana, mā reira ka whakatairanga i te horapa quinol/quinone i waenga i te RC me te matatini cytochrome bc1 (11, 13-15). Ko te tipu phototrophic papura Rhodopseudomonas (Rps.) he tauira rauropi ka taea te mārama ki te whakawhiti pūngao me te irahiko e tautoko ana i te photosynthesis. Ko te hanganga tioata tuatahi o Rps. Ko te tauira o te matatini palustris RC-LH1 ko te RC, e karapotia ana e te 15 ngā porowhita heterodimeric LH1, e whakakorea ana e tētahi pūmua kāore i te mōhiotia ko "Protein W" (14). I muri mai ka tautuhia te Protein-W ko RPA4402, he pūmua 10.5kDa kāore i te tautuhia me ngā porowhita transmembrane e toru i matapaetia (TMH) (16). E whakaaro ana mātou kia whakaingoatia anō te ira rpa4402 e whakamārama ana i te pūmua W ki te pufW kia rite ki te whakaingoatanga e whakamahia ana mō ngā ira e whakamārama ana i ngā waeine RC-L, M (pufL, pufM) me LH1α, β (pufA, pufB). He mea whakamīharo, kei roto noa i te 10% o te RC-LH1 te pūmua-W, e whakaatu ana ko Rps. palustris e rua ngā matatini RC-LH1 rerekē e puta ana. I konei, ka ripoatahia e mātou ngā hanganga cryo-EM (cryo-EM) taumira teitei o ngā matatini matua e rua, tētahi me te pūmua W me te 14 αβ heterodimers, tētahi kāore he pūmua W me te koropiko 16 Heterodimer LH1 kua kati. Ko tā mātou hanganga he huringa taahiraa i roto i te māramatanga o te matatini RC-LH1 o Rps. palustris, nā te mea kua tātarihia e mātou te taupori ōrite o ia momo, ā, he nui te taumira hei tohu mārama i ia peptide me ngā tae here me ngā lipid me ngā quinones e pā ana. Mā te whakataurite i ēnei hanganga e whakaatu ana ko ngā pūmua TMH-W e toru kāore anō kia kitea i roto i tētahi atu matatini RC-LH1 tae noa mai ki tēnei wā ka whakaputa i tētahi hongere quinone hei whakatere i te whakawhitiwhiti quinone/quinolone. He maha ngā pae herenga lipid me te quinone kua tiakina kua tautuhia, ā, kua whakakitea e mātou he huringa āhua hou i muri i te whakakotahitanga o te quinone me te RC, e tika ana pea mō te pūnaha whakaahua II (PSII) RC o ngā rauropi phototrophic whai hāora. Mā ā mātou kitenga ka whakaratohia he māramatanga hou ki te nekehanga o te herenga me te whakawhitiwhiti quinone/quinolone i roto i te matatini matua RC-LH1 o ngā huakita phototrophic papura.
Hei whakahaere i tētahi rangahau taipitopito mō ngā matatini e rua i kitea i roto i a Rps. palustris, ka wehea e mātou ia RC-LH1 mā ngā tikanga matū koiora. I purea te matatini ngoikore-pūmua W (e kiia nei i muri nei ko ΔpufW) mai i te momo kāore i te ira pufW (16), ā, kotahi anake te matatini RC-LH1 ka taea te whakaputa. Ko te matatini kei roto te pūmua W ka whakaputaina e tētahi momo. Ka whakarerekētia te pūmua W o tēnei momo ki tētahi tohu 10x His i tōna pito-C, kia taea ai te whakakotahi pai i te matatini kei roto te pūmua W me te nuinga o te pūmua W kāore i te ngaro mā te aukati i te konganuku. Ka wehea pai te matatini (16) Affinity Chromatography (IMAC).
E ai ki te Pikitia 1, kei roto i ngā matatini e rua he wae-iti e toru o te RC (RC-L, RC-M me te RC-H) e karapotia ana e te pūihi LH1. Ko te hanganga 2.80-A o te matatini kāore he pūmua-W e whakaatu ana i te 16 αβ heterodimers, e hanga ana i tētahi koropiko LH1 kati e karapoti katoa ana i te RC, e kiia nei i muri nei ko te matatini RC-LH116. Ko te hanganga 2.65Å o te matatini kei roto te pūmua-W he 14-heterodimer LH1 e haukotihia ana e te pūmua-W, e kiia nei i muri nei ko te RC-LH114-W.
(A me te B) Te whakaaturanga o te mata o te pūhui. (C me te D) Ngā tae here e whakaatuhia ana i roto i ngā tokotoko. (E me te F) Ko ngā matatini i kitea mai i te mata cytoplasmic kei roto ngā peptide me ngā waeine LH1 e whakaatuhia ana i roto i ngā pakiwaituhi, ā, kua tauhia ki te taha matau mai i te āputa pūmua-W [e rite ana ki te tau Rba. matatini sphaeroides (13)]. Mō te LH1-α, ko te tae o te waeine pūmua he kōwhai; mō te LH1-β, ko te tae o te waeine pūmua he kikorangi; mō te pūmua-W, he whero te pūmua; mō te RC-H, he kikorangi; mō te RC-L, he karaka; mō te RC-M, he magenta. Ko ngā taunga honohono e tohuhia ana e ngā tokotoko, ko te kakariki e tohu ana i ngā ngota BChl me te BPh a, ko te papura e tohu ana i ngā carotenoids, ā, ko te kōwhai e tohu ana i ngā ngota UQ10. (G me te H) Tirohanga whakanuia o te āputa pūmua-W i te rohe ōrite o te matatini RC-LH114-W (G) me te matatini RC-LH116 (H). Ka whakaatuhia ngā tauhohenga ā-pūhui i te āhua o te whakakī ātea, ka whakaatuhia te quinone chelated i te kikorangi. Ka whakamāramahia te āputa pūmua-W e te rārangi pīrangi kikorangi i (G), ā, ko ngā kōhao iti e horapa ana te quinone/quinolol i runga i te mowhiti LH116 ka whakamāramahia e te rārangi pīrangi pango i (H).
E whakaatu ana te Pikitia 1 (A me B) i te RC e karapotia ana e ngā rarangi tuwhera, kati rānei o ngā heterodimers LH1αβ, e herea ana e ia BChl e rua me te carotenoid kotahi (Pikitia 1, C me D). Kua whakaatuhia e ngā rangahau o mua ko Rps te matatini LH1. I roto i te ara biosynthetic o te spirulina xanthin, kei roto i ēnei momo he taupori whakauru o ngā carotenoid (17). Heoi, ko te spiropyrroxanthin te carotenoid matua, ā, he pai tōna kiato. Nō reira, i whiriwhiria e mātou te tauira spiroxanthin i ngā pae here LH1 katoa. Ko ngā polypeptides alpha me beta he TMH takitahi me ngā rohe o waho o te membrane poto (Pikitia 1, A, B, E, me F). Ahakoa kāore i kitea te kiato o ngā toenga 17 i te C-terminus, i tapahia te polypeptide alpha mai i Met1 ki Ala46 i roto i ngā matatini e rua. I whakaitihia te polypeptide β mai i Gly4 ki Tyr52 i roto i te RC-LH116, ā, mai i Ser5 ki Tyr52 i roto i te RC-LH114-W. Kāore i kitea he mātotoru o te 3, 4 rānei o ngā toenga N-terminal, 13 rānei o te C-terminal (Pikitia S1). Ko te tātari ira-papatipu o te matatini RC-LH1 whakauru i hangaia mai i te momo-mohoao i whakaatu ko te rohe ngaro ko te hua o te wehenga heterologous o ēnei peptides (Pikitia S1 me S2). I kitea anō hoki te hanganga N-terminal o te α-Met1 (f). I whakaatuhia e te tātari ko te α-peptide he mea hanga mai i ngā toenga fMet1 ki Asp42/Ala46/Ala47/Ala50, ā, ko te β-peptide he mea hanga mai i ngā toenga Ser2 ki Ala53, e rite ana ki te mahere mātotoru EM pāmahana-iti.
Mā te whakaritenga o te α-His29 me te β-His36 ka tūtaki ngā BChl ki te kanohi; ka huihui ia αβ heterodimer me ōna hoa tata hei hanga i tētahi koropiko tuwhera (RC-LH114-W) he koropiko kati rānei (RC-LH116) huri noa i te RC. Ko te rārangi tae exciton coupled (Pikitia 1, C me te D). Ki te whakatauritea ki te roopu 877 nm o te RC-LH114-W, ko te nekehanga whero mimiti 880 nm o te RC-LH116 he 3 nm (Pikitia 2A). Heoi, he tata rite te whānuitanga porowhita dichroism (Pikitia 2B), e tohu ana ahakoa he rerekētanga mārama i waenga i ngā koropiko tuwhera me ngā koropiko kati, he tino ōrite te taiao ā-rohe o ngā BChl. Ko te nekehanga whero mimiti pea te hua o te nekehanga wera iti me te pikinga o te pumau i runga i te koropiko kati (18, 19), te huringa o te hononga tae i puta mai i te koropiko kati (20, 21), te huinga rānei o ēnei pānga e rua (11).
(A) Ngā hihi mimiti ultraviolet/kitea/tata-infrared, ko ngā tihi o ēnei kua tohua ki ō rātou tae e rite ana, ā, kua whakatauritea ki te tihi BPh i te 775 nm. (B) Ngā hihi dichroism porowhita kua whakatauritea ki te mimiti BChl i te 805 nm. (C me te D) Ngā hihi ΔA kua tīpakohia mai i ngā hihi mimiti kua whakatauhia i te wā o te matatini RC-LH114-W (C) me te matatini RC-LH116 (D). Mō te whakataurite pai ake, kua whakatauritea ngā hihi katoa ki te ∆A o −A i te 0.2 ps. (E) Te tere o te waikura cytochrome c2 i muri i te whakamārama i te aroaro o ngā kukū rerekē o UQ2 (tirohia te Pikitia S8 mō ngā raraunga mata). (F) I roto i ngā pūtau i tipuhia i raro i te mārama iti, reo, teitei rānei (10, 30, 300μMm-2 s-1 rānei), ko ngā waeine pūmua W me RC-L i roto i te matatini kua purea me te ōwehenga kiriuhi kua wehea. Whakatauhia te taumata pūmua mā te SDS-polyacrylamide gel electrophoresis me te immunoassay (tirohia te Pikitia S9 mō ngā raraunga mata). Whakatauhia te ōwehenga e pā ana ki te matatini RC-LH114-W kua purea. Ko te ōwehenga stoichiometric o RC-L ki te pūmua-W o te matatini he 1:1.
Ko ngā BChl i te tūranga 1 i roto i te koropiko αβ14 kua whakarerekētia o RC-LH114-W (Pikitia 1, A, C, me E) he tata ake ki te kaituku tuatahi RC (P) mā te 6.8Å i ngā BChl ōrite i roto i te RC-LH116 (Pikitia 1, B, D, me F, me te Pikitia S3); heoi, ko te kaha mimiti tauwhiro o ngā matatini e rua e whakaatu ana mō te RC-LH114-W me te RC-LH116, ko ngā pūmau wā whakawhiti pūngao whakaohooho mai i LH1 ki RC he 40 ±4 me te 44 ±3 ps (Pikitia 2). , C me D, Pikitia S4 me te Ripanga S2). Kāore hoki he rerekētanga nui i roto i te whakawhiti hiko i roto i te RC (Pikitia S5 me te tuhinga tāpiri e pā ana). E whakapae ana mātou ko te ōritetanga tata o te wā whakawhiti pūngao i waenga i te LH1 me te RC-P nā te tawhiti, te koki me te pūngao pūmanawa ōrite o te nuinga o ngā BChl i roto i ngā koropiko LH1 e rua. Te āhua nei kāore e tere ake te tūhura i te tauira pūngao LH1 kia tae atu ki te tawhiti iti rawa atu i te whakawhiti pūngao tika mai i ngā wāhi kāore i te tino pai ki te RC. Tērā pea ka pāngia te koropiko tuwhera LH1 i roto i te RC-LH114-W e te nekehanga wera iti i raro i ngā āhuatanga pāmahana iti mō te tātari hanganga, ā, he roa ake te hanganga mowhiti αβ14 i te pāmahana rūma mai i te tawhiti tae o ngā βBChl i te tūranga o RC 1.
Kei roto i te matatini RC-LH116 e 32 BChls me te 16 carotenoids, ā, he rite tonu tōna whakaritenga whānui ki tērā i whiwhi mai i te Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), te momo Thiorhodovibrio (Trv.) 970 (PDB ID 7C9R) (12) me te rimurimu matomato (Blc.viridis) (PDB ID 6ET5) (10). I muri i te whakarārangitanga, he iti noa ngā rerekētanga i roto i ngā tūranga o ngā heterodimers αβ i kitea, inā koa ko te 1-5, 15, me te 16 (Pikitia S6). He pānga nui te aroaro o te pūmua-W ki te hanganga o te LH1. Ko ōna TMH e toru e honoa ana e ngā koropiko poto, ko te N-terminal kei te taha lumen o te matatini me te C-terminal kei te taha cytoplasmic (Pikitia 1A me te 3, A ki D). He nui te ātete wai o te Protein-W (Pikitia 3B), ā, ka taunekeneke a TMH2 me TMH3 ki a LH1αβ-14 hei hanga i tētahi mata whakawhiti-membrane (Pikitia 3, B me E ki G). Ko te nuinga o te atanga he mea tito mai i ngā toenga Phe, Leu me Val i te rohe whakawhiti-membrane. Kua whakarārangihia ēnei toenga ki ngā waikawa amino ātete wai me ngā tae αβ-14. Ka whai wāhi atu hoki ētahi toenga polar ki te taunekeneke, tae atu ki te here hauwai i waenga i a W-Thr68 me β-Trp42 i runga i te mata o te kōhao matatini (Pikitia 3, F me G). I runga i te mata o te cytoplasm, kei te taha o te rōpū keto o ngā carotenoid αβ-14 a Gln34. Hei tāpiri, kua whakakorea te ngota ngota n-dodecyl β-d-maltoside (β-DDM), ā, kua toro atu tōna hiku ātete wai ki te atanga i waenga i te protein-W me αβ-14, ā, kei roto pea i te tinana te hiku lipid. I kite anō mātou he tino tata ngā rohe whakataunga C-terminal o te pūmua W me te RCH, engari kāore i roto i te whānuitanga o te hanga taunekeneke motuhake (Pikitia 1, A me E). Heoi anō, tera pea he taunekeneke i roto i ngā waikawa amino C-terminal kāore anō kia whakatauhia o ēnei pūmua e rua, e whakarato ana pea i tētahi tikanga mō te kohikohi i te pūmua-W i te wā e huihui ana te matatini RC-LH114-W.
(A) Ko te Pūmua-W, e anga atu ana ki te atanga ki te LH1αβ14 i roto i te āhua pakiwaituhi, he mekameka taha āhua tokotoko (whero), e whakaaturia ana i roto i tētahi wāhanga o te hoahoa pūmanawa hikohiko (mata hina mārama me te taumata āhua o te 0.13). (B) Ko te Pūmua-W e tohuhia ana e te mata tae waipēhi. Ko ngā wāhi pōro me ngā wāhi kua utaina e whakaaturia ana i roto i te kikorangi, ko ngā wāhi waipēhi e whakaaturia ana i roto i te mā, ā, ko ngā wāhi waipēhi tino kaha e whakaaturia ana i roto i te karaka. (C me te D) Ko te Pūmua-W e whakaaturia ana i roto i te pakiwaituhi, ko tōna whakatakotoranga he rite tonu ki te (A) (C), ā, ka hurihia e te 180° (D). E ai ki te tūranga i roto i te raupapatanga, ka tangohia e ngā toenga wehewehe he kaupapa tae āniwaniwa, ko te pito-N he kikorangi, ā, ko te pito-C he whero. (E) Ko te Pūmua-W i roto i te tirohanga kotahi ki te (A), ā, ko ngā toenga i te atanga o te pūmua-W:LH1 e tohuhia ana e ngā tokotoko me ngā tohu piri. (F) Ka hurihia te Pūmua-W ki te 90° e pā ana ki (E) me LH1αβ14 i roto i te whakaaturanga pakiwaituhi, me te pā ana ki ngā toenga atanga i roto i te whakaaturanga pae. Kua tohua ngā toenga iri mai i te beta polypeptide. Ka whakaaturia te cofactor hei pae e ōrite ana ki te tae o te Pikitia 1, ka whakaaturia te β-DDM kua pakaru ki te hina, ā, ka whakaaturia te hāora ki te whero. (G) Ka hurihia te tirohanga i roto i te (F) ki te 180°, me ngā toenga e kitea nuitia ana o te alpha polypeptide kua tohua.
Ka whakakapia e te Protein-W tētahi heterodimer αβ (te 15 i te Pikitia 1F), ā, ka ārai i te katinga o te koropiko, ka whakatītaha hoki i ngā heterodimer αβ tuatahi e toru. I kitea ko te koki whakatītaha mōrahi o te heterodimer αβ-1 tuatahi e pā ana ki te kiriata noa he 25° ki te 29° (Pikitia 1, A me E), i hangaia e te whakatītaha 2° ki te 8° o te αβ-1 i roto i te RC A sharp contrast-LH116 (Pikitia 1, B me F). Ko ngā heterodimer tuarua me tuatoru he whakatītaha i te 12° ki te 22° me te 5° ki te 10°, ia. Nā te aukati steric o te RC, kāore te whakatītaha o te αβ-1 e whakauru i te takirua tuarua o te αβ (e rite ana ki te 16 o te αβ i te Pikitia 1F), ā, ka hangaia he āputa mārama i roto i te mowhiti LH1 (Pikitia 1, A me E). Nā te korenga o ngā heterodimer αβ e rua, me te ngaronga o ngā BChl e whā me ngā carotenoid e rua, kāore tētahi o ngā carotenoid e herea ana ki te waeine αβ-1 kua mirohia, ka hua ake he mowhiti LH114-W kei roto ko ngā carotenoid 13 Vegetarian me ngā BChl 28. Ko ngā whakatau tata o te whakataunga ā-rohe o ngā matatini e rua i roto i ngā rohe αβ1 ki te 7 he iti iho i ērā o te toenga o te koropiko LH1, e whakaata ana pea i te kirihou ā-roto o te waeine LH1 e tata ana ki te pae RC QB (Pikitia 4).
Ko ngā pikitia o RC-LH114-W (A me B) me RC-LH116 (C me D) e whakaaturia ana mai i te tirohanga o runga/tiro taha kotahi (A me B) (A me C) me te mata kōhao o te Pikitia 1. (B me D). Kei te taha matau ngā kī tae.
Ko tētahi atu matatini matua āhuatanga anake me te ōwehenga stoichiometric o te 1:14 ko te dimer Rhodococcus sphaeroides (Rba.) RC-LH1-PufX (13). Heoi, kāore he hononga ōrite e kitea ana i roto i te pūmua W me te PufX, ā, he pānga nui ki ō rāua hanganga LH1. He TMH kotahi a PufX me te rohe cytoplasmic N-terminal e taunekeneke ana ki te taha cytoplasmic o te waeine RC-H (13) i te tūranga e rite ana ki a Rps. palustris LH116αβ-16. Ka hangaia e PufX he hongere mō te whakawhitiwhiti quinone/quinolone i waenga i te RC-LH1 me te matatini cytochrome bcl, ā, kei roto i ngā matatini matua Rba. sphaeroides katoa (13). Ahakoa kei roto i te Rba te atanga monomer-monomer. Kei te tūranga here o te pūmua W i roto i te RC-LH114-W te dimer sphaeroides RC-LH1-PufX, ā, kei te tūranga ōrite te āputa i puta mai i a PufX me te pūmua-W (Pikitia S7A). Kei te hangai hoki te āputa i roto i te RC-LH114-W ki te hongere quinone whakapae (8) o Pseudomonas rosea LH1, i hangaia e ngā peptide kāore e pā ana ki te pūmua W, ki a PufX rānei (Pikitia S7B). Hei tāpiri, ko te hongere quinone i roto i te Blc. Ko te LH1 kakariki emerara i hangaia mā te tango i tētahi waeine γ (7) kei te tūranga ōrite (Pikitia S7C). Ahakoa he rerekē ngā pūmua e whakawhitiwhitihia ana, ko te āhua o ēnei hongere quinone/quinolol i roto i te tūranga kotahi i roto i te matatini RC-LH1 te āhua nei he tauira o te whanaketanga honohono, e tohu ana ko te āputa i hangaia e te pūmua W ka mahi pea hei hongere quinone.
Mā te āputa i roto i te koropiko LH114-W ka taea te hanga i tētahi rohe kiriuhi tonu i waenganui i te wāhi o roto o te matatini RC-LH114-W me te kiriuhi papatipu (Pikitia 1G), kaua e hono i ngā rohe e rua mā roto i tētahi pūwero pūmua pērā i roto i ngā pūmua. He rite te matatini RC-LH116 ki tētahi matatini Tch kua kati. He rite ki te ngira (22) (Pikitia 1H). Nā te mea he tere ake te horapa o te quinone mā roto i te kiriuhi i te horapa mā roto i te hongere pūmua whaiti, ka taea e te koropiko LH114-W tuwhera te tuku i te huringa RC tere ake i te koropiko LH116 kua kati, ā, ka iti ake pea te horapa o te quinone ki roto i te RC. Hei whakamātautau mēnā ka pā te pūmua W ki te huringa o ngā quinones mā roto i te RC, i whakahaerehia e mātou he whakamātautau whakakōkī cytochrome i runga i tētahi kukū o te ubiquinone 2 (UQ2) (he rite ki te UQ10 tūturu me te hiku isoprene poto ake) (Pikitia 2E). Ahakoa te noho mai o te quinone chelated e whakararu ana i te whakatau tika o te pūmau Michaelis e kitea ana (he pai te RC-LH114-W me te RC-LH116 mō te 0.2±0.1μM me te 0.5±0.2μM, ia), ko te tere mōrahi o te RC-LH114-W (4.6±0.2 e-RC-1 s-1) he 28±5% te nui ake i te RC-LH116 (3.6±0.1 e-RC-1 s-1).
I te tīmatanga i whakatauhia e mātou kei roto i te 10% o te matatini matua te pūmua-W (16); i konei, ko ngā reiti nohoanga o ngā pūtau tipu iti-mārama, waenga-mārama, me te mārama-nui he 15±0.6%, 11±1% me te 0.9±0.5, ia % (Pikitia 2F). I whakaatuhia e te whakataurite ine o te ine papatipu kāore te tāpiri o te tohu histidine i whakaiti i te nui o te pūmua-W ki te whakataurite ki ngā momo momo-mohoao (P = 0.59), nō reira ehara ēnei taumata i te taonga o te pūmua-W kua whakarerekētia (Pikitia S10). Heoi, ko tēnei nohoanga iti o te pūmua-W i roto i te matatini RC-LH1 ka taea e ētahi RC te huri tere, ā, ka whakaiti i te whakawhitinga quinone/quinolone puhoi i roto i te matatini RC-LH116. I kite mātou ko te reiti nohoanga teitei o te mārama kāore i te ōrite ki ngā raraunga transcriptomics hou, e tohu ana ka piki ake te whakaaturanga ira pufW i raro i te mārama kaha (Pikitia S11) (23). He rangirua te rerekētanga i waenga i te tuhinga pufW me te whakaurunga pūmua-W ki roto i te matatini RC-LH1, ā, tera pea e whakaata ana i te whakahaere matatini o te pūmua.
I roto i te RC-LH114-W, e 6 ngā cardiolipin (CDL), e 7 ngā phosphatidylcholine (POPC), e 1 ngā phosphatidylglycerol (POPG) me te 29 ngā ngota β-DDM kua tohaina, ā, kua tauirahia ki roto - e 6 ngā CDL, e 24 ngā POPC, e 2 ngā POPG me te 12 ngā βDDM. RC-LH116 (Pikitia 5, A me B). I roto i ēnei hanganga e rua, kei te taha cytoplasmic o te matatini te CDL, ko te nuinga ia o te POPC, te POPG me te β-DDM kei te taha luminal. E rua ngā ngota lipid me te hopi i wehea i te rohe αβ-1 ki te αβ-6 o te matatini RC-LH114-W (Pikitia 5A), ā, e rima i wehea i te rohe ōrite o RC-LH116 (Pikitia 5B). I kitea ētahi atu ngako i tērā taha o te matatini, ko te nuinga ko te CDL, i kohia i waenga i te RC me te αβ-7 ki te αβ-13 (Pikitia 5, A me te B). Kei waho o te mowhiti LH1 ētahi atu ngako me ngā hopi horoi kua taurite te hanganga, ā, ka toro atu ngā mekameka acyl kua taurite te hanganga i waenga i ngā waeine LH1, e tohua ana ko te β-DDM i roto i te RC-LH114-W, ā, kua tautuhia ko te β-DDM i roto i te ranunga RC A o te β-DDM me te POPC-LH116. Ko ngā tūranga ōrite o ngā ngako me ngā hopi horoi chelating i roto i tō tātou hanganga e tohu ana he pae here e tika ana mō te tinana (Pikitia S12A). He pai hoki te ōrite o ngā tūranga o ngā ngota ngota ōrite i roto i te Tch. Gentle me Trv. Ko te momo 970 RC-LH1s (Pikitia S12, B ki E) (9, 12) me ngā toenga here-hauwai o te rōpū pane ngako i whakaatu i te tiaki pai i roto i te whakaritenga raupapa (Pikitia S13), e tohu ana ko te CDL Tiaki e herea ana ki te RC (24), ka tiakina pea ēnei pae i roto i te matatini RC-LH1.
(A me B) Ko ngā peptide RC-LH114-W (A) me RC-LH116 (B) e whakaatuhia ana e ngā pakiwaituhi, ā, ko ngā tae e whakaatuhia ana e ngā tokotoko, mā te whakamahi i te kaupapa tae i te Pikitia 1. E whakaaturia ana ngā lipid i te whero, ā, e whakaaturia ana ngā hopi horoi i te hina. Ko te UQ e herea ana ki ngā pae RC QA me QB he kōwhai, ko te UQ wehea he kikorangi. (C me D) Ko ngā tirohanga ōrite ki (A) me (B), me ngā lipid kua tangohia. (E ki G) Tirohanga whakanuia o Q1(E), Q2(F) me Q3(G) mai i te RC-LH116, me ngā mekameka taha e awe ana tetahi ki tetahi. Ko ngā here hauwai e whakaatuhia ana hei rārangi pango kua pīrangihia.
I roto i te RC-LH116, ka wehea te RC QA me te QB UQ, e whai wāhi ana ki te whakawhiti irahiko i roto i te tukanga wehenga utu, i roto i ō rāua pae here. Heoi, i roto i te RC-LH114-W, kāore anō kia whakatauhia te quinone QB, ā, ka matapakihia taipitopito i raro nei. Haunga ngā quinones QA me QB, e rua ngā ngota UQ chelated (kei waenganui i ngā mowhiti RC me LH1) kua tohaina ki roto i te hanganga RC-LH114-W e ai ki ō rāua rōpū upoko kua whakatauhia pai (kei roto i te Q1 me te Q2, ia). wāhi). Pikitia 5C). E rua ngā wae isoprene kua tohaina ki te Q1, ā, ka whakatauhia e te mahere kiato ngā hiku isoprene 10 katoa o te Q2. I roto i te hanganga o te RC-LH116, e toru ngā ngota UQ10 chelated (Q1 ki te Q3, Pikitia 5D) i whakatauhia, ā, he mārama te kiato o ngā ngota katoa puta noa i te hiku (Pikitia 5, D ki te G). I roto i ngā hanganga e rua, he tino ōrite ngā tūranga o ngā rōpū pane quinone o Q1 me Q2 (Pikitia S12F), ā, ka taunekeneke noa rātou ki te RC. Kei te tomokanga o te āputa W o RC-LH114-W te Q1 (Pikitia 1G me te 5, C, D me E), ā, kei te taha tata o te pae here QB te Q2 (Pikitia 5, C, D) me F). He tino tata ngā toenga L-Trp143 me L-Trp269 kua tiakina ki te Q1 me Q2, ā, ka whakaratohia he taunekeneke pea o te π-stacking (Pikitia 5, E me F, me te Pikitia S12). Ko te L-Gln88, 3.0 Å mai i te hāora tawhiti o Q1, ka whakaratohia he here hauwai kaha (Pikitia 5E); ka tiakina tēnei toenga i roto i ngā RC katoa engari ko te whanaungatanga tawhiti rawa (Pikitia S13). Ko te L-Ser91 te whakakapinga ā-pūmau mō te Thr i roto i te nuinga o ngā RC kē atu (Pikitia S13), he 3.8 Angstroms mai i te hāora methyl o Q1, ā, tera pea ka whakarato i ngā here hauwai ngoikore (Pikitia 5E). Te āhua nei kāore he taunekeneke motuhake a Q3, engari kei roto i te rohe hydrophobic i waenganui i te waeine RC-M me te waeine LH1-α 5 ki te 6 (Pikitia 5, D me G). Kua whakatauhia hoki a Q1, Q2 me Q3, ngā quinones chelated tata rānei i roto i a Tch. Gentle, Trv. Strain 970 me Blc. Ko te hanganga iris (9, 10, 12) e tohu ana ki tētahi pae here quinone āwhina kua tiakina i roto i te matatini RC-LH1 (Pikitia S12G). E ōrite ana ngā UQ e rima kua pakaru i roto i te RC-LH116 ki te 5.8±0.7 o ia matatini i whakatauhia e te chromatography wai mahi teitei (HPLC), ko ngā UQ e toru kua pakaru i roto i te RC-LH114-W he iti iho i te . Ko te uara ine o te 6.2±0.3 (Pikitia S14) e tohu ana kei roto i te hanganga ngā ngota UQ kāore anō kia pakaru.
Kei roto i ngā polypeptide L me M rūpahu-āhua e rima ngā TMH, ā, ka hangaia he heterodimer e whakakotahi ana i te kotahi BChl dimer, e rua ngā monomer BChl, e rua ngā monomer bacteriophage (BPh), me te kotahi rino kore-Heme me te kotahi, e rua rānei ngā ngota UQ10. Mā te aroaro o ngā here hauwai i runga i te rōpū ketone whakamutunga me tōna kohikohinga e mōhiotia ana i roto i ngā Rp, ka whakauruhia ngā carotenoid ki roto i te waeine-M, e kiia nei ko cis-3,4-dehydroorhodopin. Momo (25). Ko te rohe kiriuhi o waho o RC-H e mau ana ki te kiriuhi e te TMH kotahi. He rite te hanganga RC whānui ki te waeine-toru RC o ngā momo e pā ana (pērā i a Rba). sphaeroides (PDB ID: 3I4D). Ko ngā huringa nui o BChl me BPh, te tuara carotenoid me te rino kore-heme e taupatupatu ana i roto i te awhe whakataunga o ēnei hanganga, pērā i te rōpū upoko UQ10 i te pae QA me te quinone QB i RC-LH116 (Pikitia S15).
Ko te wātea mai o ngā hanganga RC e rua me ngā reiti nohonga pae QB rerekē e whakarato ana i tētahi whai wāhitanga hou ki te tirotiro i ngā huringa āhua ōrite e haere tahi ana me te herenga QB quinone. I roto i te matatini RC-LH116, kei te tūranga "tata" kua herea katoatia te QB quinone (26), engari ko te wehenga o RC-LH114-W kāore he QB quinone. Kāore he QB quinone i roto i te RC-LH114-W, he mea miharo tēnei nā te mea he kaha te matatini, nui atu i te matatini RC-LH116 me te QB quinone kua whakatauhia te hanganga. Ahakoa e ono ngā quinone e hono ana ngā mowhiti LH1 e rua, e rima kua whakatauhia te hanganga i roto i te mowhiti RC-LH116 kua kati, e toru anake kua herea te hanganga i roto i te mowhiti RC-LH114-W tuwhera. Ko tēnei raruraru hanganga kua piki ake pea e whakaatu ana i te tere o te whakakapinga o ngā pae QB RC-LH114-W, te tere o te kinetics quinone i roto i te matatini, me te pikinga o te tūponotanga ki te whiti i te koropiko LH1. E kī ana mātou ko te korenga o te UQ i te pae RC QB o RC-LH114-W he hua pea nō tētahi matatini ake, ā, kua whakatio tonutia te pae QB o RC-LH114-W i roto i te huringa UQ. Ko te wāhanga motuhake (kua kati te tomokanga ki te pae QB) e whakaata ana i te āhua o tēnei mahi.
Ki te kore he QB, ka huri haere te L-Phe217 ki tētahi tūranga kāore e hototahi ki te herenga UQ10, nā te mea ka puta he tukinga ā-wāhi ki te wae isoprene tuatahi o te hiku (Pikitia 6A). Hei tāpiri, he mārama ngā huringa āhua matua, inā koa te helix de (helix poto i roto i te koropiko i waenga i te TMH D me te E) kei reira te L-Phe217 e nekehia ana ki te pute herenga QB me te hurihanga o te L-Tyr223 (Pikitia 6A) Hei wawahi i te here hauwai ki te anga M-Asp45 me te kati i te tomokanga o te pae herenga QB (Pikitia 6B). Ka huri te helix de i tōna turanga, ka nekehia te Cα o L-Ser209 mā te 0.33Å, ko te L-Val221Cα e nekehia ana mā te 3.52Å. Kāore he huringa e kitea ana i roto i te TMH D me te E, e taea ana te tāpiri i roto i ngā hanganga e rua (Pikitia 6A). E ai ki tā mātou e mōhio ana, koinei te hanganga tuatahi i roto i te RC tūturu e kati ana i te pae QB. Mā te whakataurite ki te hanganga katoa (e herea ana ki te QB) e whakaatu ana i mua i te whakaitinga o te quinone, me huri te āhua kia uru atu ai ki te quinone. Ka huri a L-Phe217 ki te hanga i tētahi taunekeneke π-stacking me te rōpū pane quinone, ā, ka neke te helix ki waho, ka taea ai e te angaanga o L-Gly222 me te mekameka taha o L-Tyr223 te hanga i tētahi whatunga here hauwai me tētahi hanganga here hauwai pumau (Pikitia 6, A me C).
(A) He pakiwaituhi taupatupatu o te hanganga hologram (mekameka L, karaka/mekameka M, magenta) me te apo (hina), e whakaatuhia ana ngā toenga matua i roto i te āhua o te whakaaturanga tokotoko. Ko te UQ10 e tohuhia ana e te pae kōwhai. Ko te rārangi iraira e tohu ana i ngā here hauwai i hangaia i roto i te hanganga katoa. (B me te C) Ko te whakaaturanga mata o te apolipoprotein me te hanganga mowhiti katoa, e whakaatu ana i te hāora mekameka taha o te L-Phe217 i te kikorangi me te L-Tyr223 i te whero. He karaka te waeine L; kāore i te tae ngā waeine M me te H. (D me te E) Apolipoprotein (D) me te katoa (E) ngā pae RC QB [taehia e (A) ia] me Thermophilus thermophilus PSII (kakariki, kikorangi me te quinone kirihou; PDB ID: 3WU2) Whakarārangi (58).
Ahakoa he maha ngā hanganga o ngā RC kāore i te whai QB, kāore he LH1, kāore anō kia pūrongohia i mua ngā huringa āhua i kitea i roto i tēnei rangahau. Kei roto i ēnei ko te hanganga whakaheke QB mai i Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) me Rba. sphaeroides (PDB ID: 1OGV) (29), he tata rite katoa ki tō rātou hanganga QB whānui. I te tirotiro tata o te 3PRC i kitea ko ngā ngota hopi LDAO (Lauryl Dimethyl Amine Oxide) e herea ana i te tomokanga o te tūranga QB, ka ārai pea i te whakaritenga anō ki tētahi āhua kati. Ahakoa kāore te LDAO e pirau i te tūranga kotahi i roto i te 1EYS, i te 1OGV rānei, ka whakaritea ēnei RC mā te whakamahi i te hopi kotahi, nō reira ka puta pea te pānga kotahi. Ko te hanganga tioata o Rba. Ko te Sphaeroides RC i whakakotahihia ki te cytochrome c2 (PDB ID: 1L9B) te āhua nei he pae QB kua kati. Heoi, i tēnei take, ko te rohe N-terminal o te polypeptide RC-M (e taunekeneke ana ki te pae here QB mā te here H o te toenga Tyr i runga i te Q helix) ka tango i tētahi āhua koretake, ā, kāore i te tūhuratia te huringa āhua QB (30). Ko te mea e whakamarie ana, kāore anō kia kitea tēnei momo whakarerekētanga o te polypeptide M i roto i te hanganga RC-LH114-W, he tata rite ki te rohe N-terminal o te RC-LH116 RC. Me mōhio anō hoki i muri i te whakakorenga o te antenna LH1 e hangai ana ki te hopi horoi, i whakatauhia ngā apolipoprotein RC i roto i te PDB, i whakakorea ai ngā puna quinone ā-roto me ngā lipid i roto i te āputa i waenga i te RC me te mata o roto o te mowhiti LH1 e karapoti ana (31, 32). Ka noho tonu te mahi a te RC nā te mea ka mau tonu ngā kaitāpiri katoa, engari ko te quinone QB pirau, he iti ake te pumau, ā, ka ngaro pinepine i te wā o te tukanga whakarite (33). Hei tāpiri, e mōhiotia ana ko te tango i te LH1 me ngā lipid huringa taiao mai i te RC ka pā ki ngā mahi, pērā i te poto o te roa o te oranga o te āhua P+QB kua wehea e te utu (31, 34, 35). Nō reira, e whakapae ana mātou ko te noho o te mowhiti LH1 ā-rohe e karapoti ana i te RC ka pupuri pea i te pae QB "kati", ā, ka tiakina te taiao ā-rohe e tata ana ki te QB.
Ahakoa ko te apolipoprotein (kāore he QB quinone) me te hanganga katoa e tohu ana i ngā whakaahua e rua o te huringa o te pae QB, kaua i te raupapa o ngā kaupapa, kei reira ngā tohu e taea ai te herea kia kore ai e herea anō e te hydroquinone Hei aukati i te aukati substrate. Tērā pea he rerekē te taunekeneke o te quinolol me te quinone e tata ana ki te pae QB o te apolipoprotein, ka arahina ki tōna whakakorenga e te RC. Kua roa te whakaaro he wāhi nui ngā huringa āhua ki te herea me te whakaiti i ngā quinones. Kua ngoikore te kaha o ngā RC tio ki te whakaiti i ngā quinones i muri i te urutau pōuri (36); E whakaatu ana te crystallography hihi-X ko tēnei kino nā te mau o ngā quinones QB i roto i te āhua "tawhiti" tata ki te 4.5 Å mai i te tūranga tata hohe (26), 37). E kī ana mātou ko tēnei āhua here tawhiti he whakaahua o te āhua waenga i waenga i te apolipoprotein me te hanganga mowhiti katoa, e whai ake nei i te taunekeneke tuatahi me te quinone me te whakatuwheratanga o te pae QB.
Ko te momo II RC i kitea i roto i te matatini PSII o ētahi huakita phototrophic me ngā cyanobacteria, ngā rimurimu me ngā tipu he tiaki hanganga me te tiaki mahi (38). Ko te whakaritenga hanganga e whakaaturia ana i te Pikitia 6 (D me te E) e whakaatu ana i te ōrite i waenga i ngā PSII RC me te pae QB o te matatini RC huakita. Kua roa tēnei whakataurite hei tauira mō te ako i ngā pūnaha e pā tata ana ki te here me te whakaiti quinone. I kī ngā tuhinga o mua ko ngā huringa āhua e haere tahi ana me te whakaiti PSII o ngā quinones (39, 40). Nō reira, i te whakaaro ki te tiaki whanaketanga o te RC, ka taea hoki te whakamahi i tēnei tikanga here kāore i kitea i mua ki te pae QB o te PSII RC i roto i ngā tipu phototrophic oxygenated.
Ko ngā momo Rps ΔpufW (te mukunga pufW kāore i te tapaina) me PufW-His (C-terminal 10x His-tagged protein-W i whakaatuhia mai i te pufW locus tūturu). I whakaahuahia te palustris CGA009 i roto i tā mātou mahi o mua (16). I tangohia mai ēnei momo me te matua momo-mohoao isogenic mai i te pouaka tio mā te whakatō i tētahi maha iti o ngā pūtau ki runga i te PYE (ia 5 karamu rita -1) (i rongoatia ki roto i te LB i te -80 °C, kei roto ko te 50% (w/v) glycerol) pūmua, te tango rewena me te succinate) agar [1.5% (w/v)] pereti. I whaowhia te pereti i te pō i roto i te pōuri i te pāmahana rūma i raro i ngā āhuatanga anaerobic, kātahi ka whakamāramahia ki te mārama mā (~50 μmolm-2 s-1) i whakaratohia e ngā pūangi halogen OSRAM 116-W (RS Components, UK) mō ngā rā 3 ki te 5 tae noa ki te putanga mai o tētahi koroni kotahi. I whakamahia he koroni kotahi hei werowero i te 10 ml o te reo M22+ (41) i tāpirihia ki te 0.1% (w/v) waikawa casamino (e kiia nei ko M22). I whakatipuria te ahurea i raro i ngā āhuatanga hāora iti i roto i te pōuri i te 34°C me te rūrū i te 180 rpm mō ngā hāora 48, kātahi ka werowerohia te 70 ml o te ahurea i raro i ngā āhuatanga ōrite mō ngā hāora 24. I whakamahia he ahurea haurua-aerobic me te rōrahi o te 1 ml hei werowero i te 30 ml o te reo M22 ki roto i te pounamu karāhe mārama whānui 30 ml, ā, i whakamāramahia ki te whakaoho (~50μmolm-2 s-1) mō ngā hāora 48 mā te kaha aukume parakore. Kātahi ka werowerohia te 30 ml o te ahurea ki te 1 rita o te ahurea i raro i ngā āhuatanga ōrite, ā, i whakamahia hei werowero i te 9 rita o te ahurea i whakamāramahia i te ~200 μmolm-2 s-1 mō ngā hāora 72. I hauhaketia ngā pūtau mā te centrifugation i te 7132 RCF mō te 30 meneti, kātahi ka tāpirihia anō ki roto i te ~10 ml o te 20 mM tris-HCl (pH 8.0), ā, i rongoatia ki te -20°C kia hiahiatia rā anō.
I muri i te whakarewa, tāpirihia ētahi tioata o te deoxyribonuclease I (Merck, UK), te lysozyme (Merck, UK) me ngā papa aukati protease holoenzyme Roche e rua (Merck, UK) ki ngā pūtau kua whakatārewatia anō. I roto i te pūtau pēhanga French 20,000 psi (Aminco, USA), i whakararuraruhia ngā pūtau e 8 ki te 12 ngā wā. I muri i te tango i ngā pūtau kāore i pakaru me ngā para kore-rewa mā te centrifugation i te 18,500 RCF mō te 15 meneti i te 4°C, i whakapūpūhia te kiriuhi mai i te lysate tae mā te centrifugation i te 113,000 RCF mō te 2 hāora i te 43,000°C. Maka atu te wāhanga wairewa ka whakatārewa anō i te kiriuhi tae ki roto i te 100 ki te 200 ml o te 20 mM tris-HCl (pH 8.0) ka whakaranua kia kore ra ano he puranga e kitea. I whaowhia te kiriuhi iri ki roto i te 20 mM tris-HCl (pH 8.0) (Anatrace, USA) kei roto ko te 2% (w/v) β-DDM mō te 1 hāora i te pōuri i te 4°C me te whakaoho mārie. Kātahi ka whakapūtāhia i te 70°C hei whakarewa i te 150,000 RCF i te 4°C mō te 1 hāora hei tango i ngā toenga kore-rewa.
I pania te kiriuhi whakarewa mai i te momo ΔpufW ki tētahi pou whakawhiti katote DEAE Sepharose 50 ml me ngā rōrahi pou e toru (CV) o te pūmau here [20 mM tris-HCl (pH 8.0) kei roto ko te 0.03% (w/v) β-DDM]. Horoia te pou ki ngā pūmau here CV e rua, kātahi ka horoia te pou ki ngā pūmau here e rua kei roto ko te 50 mM NaCl. I whakawāteahia te matatini RC-LH116 me te rōnaki rārangi o te 150 ki te 300 mM NaCl (i roto i te pūmau here) i runga i te 1.75 CV, ā, i whakawāteahia te matatini here e toe ana ki tētahi pūmau here kei roto ko te 300 mM NaCl i runga i te 0.5 CV. Kohia te whānuitanga mimiti i waenga i te 250 me te 1000 nm, kia mau tonu te wāhanga me te ōwehenga mimiti (A880/A280) neke atu i te 1 i te 880 ki te 280 nm, whakaranua kia rua ngā wā i roto i te arai here, ā, whakamahia anō te tukanga kotahi i runga i te pou DEAE I te wā e purea ana. Whakaranua ngā wāhanga me ngā ōwehenga A880/A280 neke atu i te 1.7 me ngā ōwehenga A880/A805 neke atu i te 3.0, mahia te tuatoru o ngā huringa o te whakawhiti katote, ā, puritia ngā wāhanga me ngā ōwehenga A880/A280 neke atu i te 2.2 me ngā ōwehenga A880/A805 neke atu i te 5.0. I whakatōtōhia te matatini kua purea ā-wāhanga ki te ~2 ml i roto i te tātari porowhita Amicon 100,000 molecular weight cut-off (MWCO) (Merck, UK), ā, i utaina ki runga i te pou aukati rahi Superdex 200 16/600 (GE Healthcare, US) kei roto ko te 200 mM NaCl buffer, kātahi ka whakawāteahia ki roto i te buffer kotahi i te 1.5 CV. Kohia ngā ira mimiti o te wāhanga aukati rahi, ka whakatōtōhia ngā ira mimiti me ngā ōwehenga A880/A280 neke atu i te 2.4 me ngā ōwehenga A880/A805 neke atu i te 5.8 ki te 100 A880, ā, whakamahia tonutia mō te whakarite, te rokiroki rānei i te whatunga cryo-TEM. Puritia ki te -80°C kia hiahiatia rā anō.
I pania te kiriuhi whakarewa mai i te momo PufW-His ki te pou HisPrep FF Ni-NTA Sepharose 20 ml (20 mM tris-HCl (pH 8.0) kei roto ko te 200 mM NaCl me te 0.03% (w/w)) i roto i te pūwero IMAC (GE Healthcare). v) β-DDM]. I horoia te pou ki ngā CV e rima o te pūwero IMAC, kātahi ki ngā CV e rima o te pūwero IMAC kei roto ko te 10 mM histidine. I tangohia te matatini matua mai i te pou me ngā pūwero IMAC e rima kei roto ko te 100 mM histidine. Ko te wāhanga kei roto ko te matatini RC-LH114-W ka whakawhāititia ki te ~10 ml i roto i te tāke whakaoho me te tātari Amicon 100,000 MWCO (Merck, UK), ka waimeha kia 20 ngā wā ki te pūwero here, kātahi ka tāpirihia ki te 25 ml. I roto i te pou DEAE Sepharose, e whā ngā CV e herea ana ki te pūwero ka whakamahia i mua. Horoia te pou ki ngā arai here CV e whā, kātahi ka whakamararatia te matatini ki ngā CV e waru i runga i te rōnaki rārangi o te 0 ki te 100 mM NaCl (i roto i te arai here), me ngā CV e whā e toe ana kei roto he arai here 100 mM. Ko ngā matatini toenga i whakamararatia ki runga i te konutai pūhaumāota i honoa ki te ōwehenga A880/A280 teitei ake i te 2.4 me te ōwehenga A880/A805 teitei ake i te 4.6 i whakatōkia ki te ~2 ml i roto i te tātari porowhita Amicon 100,000 MWCO, ā, i whakakīia ki te 1.5 CV IMAC i mua. I whakatauritehia e te arai te pou aukati Superdex 200 16/600 te rahi, kātahi ka whakamararatia ki roto i te arai kotahi ki runga i te 1.5 CV. Kohia ngā ira mimiti o ngā wāhanga rahi-kore, ka whakatōkia ngā ira mimiti me ngā ōwehenga A880/A280 neke atu i te 2.1 me ngā ōwehenga A880/A805 neke atu i te 4.6 ki te 100 A880, ka whakamahia tonutia mō te whakarite i te whatunga TEM tio, ka rongoatia rānei ki te -80°C kia hiahiatia rā anō.
I whakamahia he pouaka tio rumaki Leica EM GP hei whakarite i ngā whatunga TEM pāmahana iti. I whakaranua te matatini ki te pūwero IMAC ki te A880 o te 50, kātahi ka utaina he 5μl ki runga i te whatunga parahi kua pania ki te waro QUANTIFOIL 1.2/1.3 kua mura ake (Agar Scientific, UK). Whakatōkia te whatunga ki te 20°C me te haumākū whanaunga 60% mō te 30 hēkona, kātahi ka murua kia maroke mō te 3 hēkona, kātahi ka tineia ki te wai ethane i te -176°C.
I tuhia ngā raraunga o te matatini RC-LH114-W ki te eBIC (Electronic Bioimaging Center) (British Diamond Light Source) me te karu hiko Titan Krios, e mahi ana i te ngaohiko tere o te 300kV, me te whakanui ingoa o te 130,000× me te pūngao o- Kōwhiria he āputa o te 20 eV. I whakamahia he Gatan 968 GIF Quantum me te pūoko tihi K2 hei tuhi whakaahua i te aratau tatau hei kohi raraunga. Ko te rahi pika kua whakatikatikaina ko te 1.048Å, ā, ko te tere horopeta ko te 3.83 e-Å-2s-1. I kohia te kiriata i roto i te 11 hēkona ka wehea kia 40 ngā wāhanga. Whakamahia te wāhi kua pania ki te waro hei arotahi anō i te karu hiko, kātahi ka kohia kia toru ngā kiriata mō ia kōhao. I te katoa, 3130 ngā kiriata i kohia, me ngā uara whakakore arotahi i waenga i te -1 me te -3μm.
I kohia ngā raraunga mō te matatini RC-LH116 mā te whakamahi i te karuārai kotahi i te Asterbury Biostructure Laboratory (Te Whare Wānanga o Leeds, UK). I kohia ngā raraunga i roto i te aratau tatau me te whakanui o te 130 k, ā, i whakatikatikaina te rahi o te pika ki te 1.065 Å me te horopeta o te 4.6 e-Å-2s-1. I hopukina te kiriata i roto i te 12 hēkona, ā, i wehea kia 48 ngā wāhanga. I te katoa, 3359 ngā kiriata i kohia, me ngā uara defocus i waenga i te -1 me te -3μm.
Ka mahia ngā tukatuka raraunga katoa i roto i te paipa Relion 3.0 (42). Whakamahia te Motioncorr 2 (43) hei whakatika i te nekehanga hihi mā te taumaha horopeta, kātahi ka whakamahia te CTFFIND 4.1 (44) hei whakatau i te tawhā CTF (mahi whakawhiti rerekētanga). Ko ngā whakaahua-iti noa i muri i ēnei wāhanga tukatuka tuatahi e whakaaturia ana i te Pikitia 2. S16. Ka hangaia te tauira kōwhiringa aunoa mā te tīpako ā-ringa i te 250 pika o te 1000 matūriki i roto i te anga 250-pika me te kore he whakarōpūtanga rua-ahu tohutoro (2D), nā reira ka whakakorehia aua whakarōpūtanga e tutuki ana i te poke tauira, kāore rānei he āhuatanga e kitea ana. Kātahi ka mahia te kōwhiringa aunoa ki ngā whakaahua-iti katoa, ā, ko te RC-LH114-W he 849,359 matūriki, ā, ko te matatini RC-LH116 he 476,547 matūriki. Kua rua ngā wāhanga o te whakarōpūtanga 2D kore-tohutoro i ngā matūriki katoa kua tīpakohia, ā, i muri i ia oma, ka whakakahoretia ngā matūriki e tūtaki ana ki te horahanga waro, te poke tauira, te kore āhuatanga kitea, ngā matūriki taupatupatu kaha rānei, ā, ka hua ake ko te 772,033 (90.9%) me te 359,678 (75.5%) ) ngā matūriki e whakamahia ana mō te whakarōpūtanga 3D o RC-LH114-W me RC-LH116. I hangaia te tauira tohutoro 3D tuatahi mā te whakamahi i te tikanga hekenga gradient stochastic. Mā te whakamahi i te tauira tuatahi hei tohutoro, ka whakarōpūhia ngā matūriki kua tīpakohia ki ngā kāwai e whā i roto i te 3D. Mā te whakamahi i te tauira i roto i tēnei kāwai hei tohutoro, mahia te whakapai 3D i runga i ngā matūriki i roto i te kāwai nui rawa atu, kātahi ka whakamahia te tātari iti-haere tuatahi 15Å hei taupoki i te horahanga whakarewa, tāpirihia he 6 pika o ngā taha ngohengohe, ā, tukatukahia ngā pika i muri i te tukatuka hei whakatika i te mahi whakawhiti Gatan K2 peak Modulation o te pūoko o runga. Mō te huinga raraunga RC-LH114-W, i whakarerekētia tēnei tauira tuatahi mā te tango i te mātotoru kaha i ngā taha o te kopare (i motuhia mai i te mātotoru matatini matua i roto i te UCSF Chimera). Ko ngā tauira hua (ko ngā taumira o RC-LH114-W me RC-LH116 he 3.91 me te 4.16 Å, ia) ka whakamahia hei tohutoro mō te tuarua o ngā rauna o te whakarōpūtanga 3D. Ka whakarōpūhia ngā matūriki i whakamahia ki roto i te akomanga 3D tuatahi, ā, kāore he hononga kaha ki te taha tata. Taupoki, kāore rānei he āhuatanga hanganga mārama. I muri i te tuarua o ngā rauna o te whakarōpūtanga 3D, i tīpakohia te kāwai me te taumira teitei rawa [Mō RC-LH114-W, kotahi te kāwai he 377,703 ngā matūriki (44.5%), mō RC-LH116, e rua ngā kāwai, ko te katoa he 260,752 ngā matūriki (54.7%), he rite tonu ina whakaritea i muri i te hurihanga tuatahi me te rerekētanga iti]. Ka tangohia anō ngā matūriki kua tīpakohia ki roto i tētahi pouaka 400-pika, ā, ka whakatikatikaina mā te whakatikatika 3D. Ka hangaia te kopare whakarewa mā te whakamahi i te tātari iti-haere tuatahi 15Å, te whakawhānui mahere 3 pika me te kopare ngohengohe 3 pika. Mā te whakamahi i te whakatikatika CTF mō ia matūriki, te whakatikatika nekehanga mō ia matūriki me te wāhanga tuarua o te whakatikatika CTF mō ia matūriki, te whakatikatika 3D, te kopare whakarewa me te tukatuka-muri ka mahia i muri i ia taahiraa hei whakatikatika anō i te kakano hua. Mā te whakamahi i te uara tapahi FSC (Fourier Shell Correlation Coefficient) o 0.143, ko ngā whakataunga o ngā tauira whakamutunga o RC-LH114-W me RC-LH116 he 2.65 me te 2.80Å. Kei te whakaatuhia te pihi FSC o te tauira whakamutunga i te Pikitia 2. S17.
Kua tangohia ngā raupapatanga pūmua katoa mai 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). I whakamahia te SWISS-MODEL (45) hei hanga i tētahi tauira homology o RC, kei roto ko ngā raupapatanga pūmua o RC-L, RC-M me RC-H me te hanganga tioata o Rba. I whakamahia te sphaeroides RC hei tauira (PDB ID: 5LSE) (46). Whakamahia te taputapu "mahere whakauru" i roto i te UCSF Chimera hei whakauru i te tauira kua hangaia ki te mahere (47), whakapai ake i te hanganga pūmua, me te cofactor [4×BChl a (ingoa toenga whare pukapuka monomer = BCL), 2×BPh a (BPH), kotahi, e rua rānei ngā momo UQ10 (U10), kotahi te rino kore-heme (Fe) me tetahi 3,4-dihydrohexacarbonylcholine (QAK)] whakamahia te Coot (48) hei tāpiri. Nā te mea kāore e wātea ana te QAK i roto i te whare pukapuka monomer, i whakapūtāhia mā te whakamahi i te taputapu eLBOW i roto i te PHENIX (49).
I muri mai, i hangaia te waeine LH1. I te tīmatanga, i whakamahia te taputapu hanga aunoa i roto i te PHENIX (49) hei hanga aunoa i tētahi wāhanga o te raupapatanga LH1 mā te whakamahi i te mahere me ngā raupapatanga pūmua LH1-α me te LH1-β hei whakauru. Tīpakohia te waeine LH1 tino oti, tangohia mai, ka utaina ki roto i te Coot, tāpirihia ā-ringa te raupapatanga ngaro ki roto, ka whakatikatika ā-ringa i te hanganga katoa i mua i te tāpiri i ngā BCl e rua (BCL) me te spirilloxanthin (CRT) [e ai ki ngā Rps e tika ana Te mātotoru o te matatini LH1 me te ihirangi carotenoid e mōhiotia ana. Momo (17)]. Tāruatia te waeine LH1 katoa, ka whakamahi i te UCSF Chimera "Docking Map Tool" hei taunga ki te rohe kore-tauira tata o te mātotoru LH1, kātahi ka whakatikatika i roto i te Coot; whakahokia te tukanga kia oti rā anō te whakatauira i ngā waeine LH1 katoa. Mō te hanganga RC-LH114-W, mā te tango i te mātotoru kāore i tohaina i roto i te Coot, ka wehea te pūmua mai i ngā wāhanga kore-pūmua e toe ana i roto i te mahere USCF Chimera, ā, ka whakamahia te taputapu Autobuild hei whakatū i te tauira tuatahi, me te Whakatauira i ngā waeine e toe ana (pūmua-W). I roto i te PHENIX (49). Tāpirihia ngā raupapatanga ngaro ki te tauira hua i roto i te Coot (48), kātahi ka whakatikatika ā-ringa i te waeine katoa. Ko te mātotoru kāore i tohaina e toe ana ka uru ki te huinga o ngā lipid (PDB monomer library ID o CDL = CDL, POPC = 6PL me POPG = PGT), β-DDM detergent (LMT) me ngā ngota UQ10 (U10). Whakamahia te arotautanga PHENIX (49) me te arotautanga ā-ringa i roto i te Coot (48) hei whakapai ake i te tauira tuatahi katoa tae noa ki te kore e taea te whakapai ake i ngā tatauranga tauira me te kounga tirohanga o te taunga. Hei whakamutunga, whakamahia te LocScale (50) hei whakakoi i te mahere ā-rohe, kātahi ka mahi i ētahi atu huringa o te whakatauira i te mātotoru kāore i tohaina me te arotautanga aunoa me te ā-ringa.
Ko ngā peptide, ngā cofactor me ētahi atu lipid me ngā quinone kua honoa ki roto i ō rātou ake kiato e whakaaturia ana i ngā Pikitia 1 me te 2. Mai i te Whārangi 18 ki te Whārangi 23. Ko ngā mōhiohio tatauranga o te tauira whakamutunga e whakaaturia ana i te Ripanga Whārangi 1.
Ki te kore e whakahuatia tētahi atu mea, i kohia ngā hihi mimiti UV/Vis/NIR i runga i te mita hihi Cary60 (Agilent, USA) i ngā wā 1 nm mai i te 250 nm ki te 1000 nm me te wā whakauru o te 0.1s.
Whakawaiwaihia te tauira ki roto i tētahi kuveti kuata me te ara 2 mm ki te A880 o te 1, ka kohia te whānuitanga mimiti i waenga i te 400 me te 1000 nm. I kohia ngā whānuitanga porowhita dichroic ki runga i tētahi mita spectropolari Jasco 810 (Jasco, Hapani) i ngā wā 1 nm i waenga i te 400 nm me te 950 nm i te tere matawai o te 20 nm min-1.
Ka whakatauhia te tauwehenga mōrearea molar mā te whakawaiwai i te matatini matua ki te A880 tata ki te 50. Whakawaiwaihia te rōrahi 10μl ki roto i te 990μl herenga arai, te methanol rānei, ka kohia tonutia te whānuitanga mimiti hei whakaiti i te pirau BChl. I tatauhia te ihirangi BChl o ia tauira methanol mā te tauwehenga mōrearea i te 771 nm o te 54.8 mM-1 cm-1, ā, i whakatauhia te tauwehenga mōrearea (51). Wehea te kukū BChl i inehia ki te 32 (RC-LH114-W) 36 rānei (RC-LH116) hei whakatau i te kukū matatini matua, ka whakamahia hei whakatau i te whānuitanga mimiti o te tauira kotahi i kohia i roto i te tauwehenga mōrearea arai. whakarara. E toru ngā inenga i tangohia mō ia tauira, ā, ko te mimiti toharite o te BChl Qy mōrahi i whakamahia hei tatau. Ko te tauwehenga whakamōrearea o RC-LH114-W i inehia i te 878 nm ko 3280±140 mM-1 cm-1, ko te tauwehenga whakamōrearea o RC-LH116 i inehia i te 880 nm ko 3800±30 mM-1 cm-1.
I tātaihia te UQ10 i runga anō i te tikanga i roto i te (52). I roto i te poto, i mahia te HPLC wāhanga whakamuri (RP-HPLC) mā te whakamahi i te pūnaha Agilent 1200 HPLC. Whakarewahia te 0.02 nmol o RC-LH116, RC-LH114-W rānei ki roto i te 50μl o te 50:50 methanol:chloroform kei roto ko te 0.02% (w/v) ferric chloride, ka werohia te Beckman Coulter Ultrasphere ODS 4.6 mm kua whakatauritea. Whakarewahia ki roto i te 1 ml-1 min-1 i te 40°C i roto i te whakarewa HPLC (80:20 methanol:2-propanol) i runga i te pou ×25 cm. Whakahaerehia te elution isocratic i roto i te whakarewa HPLC hei aroturuki i te mimiti i te 275 nm (UQ10), 450 nm (carotenoids) me te 780 nm (BChl) mō te 1 hāora. I whakauruhia te tihi i roto i te chromatogram 275 nm i te 25.5 meneti, kāore i roto i tēnei he pūhui kē atu ka kitea. Ka whakamahia te horahanga whakauru hei tatau i te nui molar o te UQ10 i tangohia me te tohutoro ki te pihi whakatikatika i tatauhia mai i te werohanga o ngā paerewa parakore mai i te 0 ki te 5.8 nmol (Pikitia S14). I tātarihia ia tauira i roto i ngā tāruarua e toru, ā, ko te hapa i pūrongohia e rite ana ki te SD o te toharite.
I whakaritea he otinga kei roto te matatini RC-LH1 me te mimiti Qy mōrahi o te 0.1 me te 30 μM cytochrome ngākau hōiho whakaitihia c2 (Merck, UK) me te 0 ki te 50 μMUQ2 (Merck, UK). E toru ngā tauira 1-ml i whakaritea i ia kukū UQ2, ā, i whaowhia mō te pō i roto i te pōuri i te 4°C kia tino urutau ki te pōuri i mua i te ine. I utaina te otinga ki roto i te ine matawai OLIS RSM1000 me te whatanga mura/raina 500 300 nm, te tomokanga 1.24 mm, te waenganui 0.12 mm me ngā kohao putanga 0.6 mm. Ka whakatakotoria he tātari haere 600 nm te roa ki te tomokanga o te ngongo whakaahua tauira me te ngongo whakarea whakaahua tohutoro hei aukati i te mārama whakaoho. I aroturukitia te mimiti i te 550 nm me te wā whakauru o te 0.15 s. Ka tukuna te mārama whakaohooho mai i te 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) mā roto i tētahi taura muka whatu i te kaha 90% mā roto i tētahi kaiwhakahaere DC2200 (Thorlabs Ltd., UK) ā, ka tukuna ki te pūtake mārama i te koki 90° o. Ka anga atu te hihi ine ki te whakaata hei whakahoki i tētahi mārama kāore i mimitia tuatahitia e te tauira. Aroturukihia te mimiti 10 hēkona i mua i te māramatanga o te 50 hēkona. Kātahi ka aro turukihia anō te mimiti mō te 60 hēkona i roto i te pōuri hei aromatawai i te nui o te whakaiti ā-pūnaha a te quinolol i te cytochrome c23+ (tirohia te Pikitia S8 mō ngā raraunga mata).
I tukatukahia ngā raraunga mā te whakauru i tētahi tere tīmatanga rārangi i roto i te 0.5 ki te 10 s (i runga i te kukū UQ2) me te toharite i ngā tere o ngā tauira e toru i ia kukū UQ2. Ko te kukū RC-LH1 i tatauhia e te tauwehenga whakamōkito i whakamahia hei huri i te tere ki te whai huatanga whakatere, i tuhia ki te Origin Pro 2019 (OriginLab, USA), ā, i whakaurua ki te tauira Michaelis-Menten hei whakatau i ngā uara Km me Kcat e kitea ana.
Mō ngā inenga mimiti taupua, i whakaranua te tauira RC-LH1 ki te ~2μM i roto i te pūwero IMAC kei roto ko te 50 mM konutai ascorbate (Merck, USA) me te 0.4 mM Terbutin (Merck, USA). Ka whakamahia te waikawa ascorbic hei kaituku irahiko patunga tapu, ā, ka whakamahia te tert-butaclofen hei aukati QB hei whakarite kia noho whakaitihia tonutia te kaituku RC matua (arā, kāore i te whakaahua-hauora) puta noa i te tukanga ine. Tata ki te 3 ml o te tauira ka tāpirihia ki tētahi pūtau hurihuri ritenga (tata ki te 0.1 m te whānui, 350 RPM) me te roa ara whatu 2 mm hei whakarite kia nui te wā o te tauira i roto i te ara taiaho mō te urutau pōuri i waenga i ngā pāoro whakaoho. Whakamahia ngā pāoro taiaho ~100-fs hei whakanui ake i te pūnaha taiaho Ti: Sapphire (Spectra Physics, USA) hei whakaoho i te tauira i te 880 nm i te tere whakahoki o te 1 kHz (20 nJ mō te NIR, 100 nJ rānei mō te Vis). I mua i te kohikohi raraunga, whakaaturia te tauira ki te mārama whakaoho mō te 30 meneti pea. Mā te whakaaturanga ka kore e whakahohehia te QA (tērā pea ka whakaitihia te QA kotahi, e rua rānei ngā wā). Engari kia mōhio koe ka taea te huri i tēnei tukanga nā te mea i muri i te wā roa o te urutau pōuri, ka hoki mārire te RC ki te mahi QA. I whakamahia he ine hihi Helios (Ultrafast Systems, USA) hei ine i ngā hihi whakawhiti me te wā whakaroa o -10 ki te 7000 ps. Whakamahia te pūmanawa Surface Xplorer (Ultrafast Systems, USA) hei wehewehe i ngā huinga raraunga, kātahi ka hanumi me te whakataurite. Whakamahia te mōkihi pūmanawa CarpetView (Light Conversion Ltd., Lithuania) hei whakamahi i te huinga raraunga whakakotahi hei whiwhi hihi rerekē e pā ana ki te pirau, whakamahia rānei he mahi e whakakotahi ana i ngā taupū maha me te urupare taputapu hei whakauru i te whanaketanga hihi kotahi-roa i roto i te Origin (OriginLab, USA).
E ai ki te kōrero i runga ake nei (53), i whakaritea he kiriata whakaahua kei roto he matatini LH1 kāore he pūihi RC me te pūihi LH2 ā-taha. I whakaranua te kiriuhi ki te 20 mM tris (pH 8.0) kātahi ka utaina ki roto i te cuvette quartz me te ara whatu 2 mm. I whakamahia he pahūtanga laser 30nJ hei whakaoho i te tauira i te 540 nm me te wā whakaroa o -10 ki te 7000 ps. Tukatukahia te huinga raraunga e ai ki te whakaahuatanga mō te tauira Rps. pal.
I whakapūpūhia te kiriuhi mā te whakapūpū i te 150,000 RCF mō ngā hāora e 2 i te 4°C, kātahi ka tāpirihia anō tōna mimiti i te 880 nm ki roto i te 20 mM tris-HCl (pH 8.0) me te 200 mM NaCl. Whakarewahia te kiriuhi mā te whakaoho mārire ki roto i te 2% (w/v) β-DDM mō te hāora e 1 i te pōuri i te 4°C. I whakapūpūhia te tauira ki roto i te 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) ki te kukū pūmua o te 2.5 mg ml-1 (tātaritanga Bio-Rad). I mahia he tukatuka anō mai i te tikanga kua whakaputaina i mua (54), ka tīmata ki te whakapūpū i te 50 μg pūmua ki roto i te 50 μl TEAB katoa kei roto ko te 1% (w/v) konutai laurate (Merck, UK). I muri i te sonication mō te 60 hēkona, i whakaitihia ki te 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) i te 37°C mō te 30 meneti. Mō te S-alkylation, whakatōkia te tauira ki te 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) ka tāpirihia mai i te otinga rakau isopropanol 200 mM mō te 10 meneti i te pāmahana rūma. I whakahaerehia te nakunaku proteolytic mā te tāpiri i te 2 μg ranunga trypsin/endoproteinase Lys-C (Promega UK) ā, i whakatōkia ki te 37°C mō te 3 hāora. I tangohia te surfactant laurate mā te tāpiri i te 50 μl ethyl acetate me te 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) me te vortexing mō te 60 hēkona. I whakatairangahia te wehenga ā-wāhanga mā te centrifugation i te 15,700 RCF mō te 5 meneti. E ai ki te tikanga a te kaihanga, i whakamahia he pou hurihuri C18 (Thermo Fisher Scientific, UK) hei tango mārire i te tote o te wāhanga o raro kei roto te peptide. I muri i te whakamaroke mā te centrifugation korehau, i whakarewaina te tauira ki te 0.5% TFA me te 3% acetonitrile, ā, i tātarihia te 500 ng mā te nanoflow RP chromatography me te papatipu spectrometry mā te whakamahi i ngā tawhā pūnaha kua whakamāramahia i mua.
Whakamahia te MaxQuant v.1.5.3.30 (56) mō te tautuhi me te ine i te pūmua hei rapu i te pātengi raraunga proteome Rps. palustris (www.uniprot.org/proteomes/UP000001426). Kua tukuna ngā raraunga proteomics papatipu ki te ProteomeXchange Alliance mā te putunga hoa PRIDE (http://proteomecentral.proteomexchange.org) i raro i te tohu raraunga PXD020402.
Mō te tātaritanga mā te RPLC me te electrospray ionization mass spectrometry, i whakaritea te matatini RC-LH1 mai i ngā Rps momo-mohoao. Mā te whakamahi i te tikanga i whakaputaina i mua (16), ko te kukū pūmua i hangaia i roto i ngā pūtau palustris he 2 mg ml-1 i roto i te 20 mM Hepes (pH 7.8), 100 mM NaCl me te 0.03% (w/v) β- (tātaritanga Bio-Rad) ) DDM. E ai ki te tikanga a te kaihanga, whakamahia te kete purenga 2D (GE Healthcare, USA) hei tango i te 10 μg pūmua mā te tikanga precipitation, ā, whakarewahia te precipitation ki roto i te 20 μl 60% (v/v) waikawa formic (FA), 20% (v/v) Acetonitrile me te 20% (v/v) wai. E rima ngā microliters i tātarihia mā te RPLC (Dionex RSLC) me te papatipu spectrometry (Maxis UHR-TOF, Bruker). Whakamahia te pou MabPac 1.2×100 mm (Thermo Fisher Scientific, UK) mō te wehenga i te 60°C me te 100μlmin -1, me te pikinga o te 85% (v / v) whakarewa A [0.1% (v / v) FA me te 0.02% (V/v) otinga wai TFA] ki te 85%(v/v) whakarewa B [0.1%(v/v) FA me te 0.02%(v/v) i roto i te 90%(v/v) acetonitrile TFA] Mā te whakamahi i te pūtake ionization electrospray paerewa me ngā tawhā taunoa mō te neke atu i te 60 meneti, ka whiwhi te ine papatipu i te 100 ki te 2750 m/z (ōwehenga papatipu-ki-te-utu). Mā te āwhina o te tomokanga rauemi bioinformatics ExPASy FindPept taputapu (https://web.expasy.org/findpept/), maherehia te ine papatipu ki ngā waeine o te matatini.
I whakatipuria ngā pūtau mō ngā hāora 72 i raro i te 100 ml NF-iti (10μMm-2 s-1), reo (30μMm-2 s-1) teitei rānei (300μMm-2 s-1) mārama. M22 reo (M22 reo kāore i whakaurua te ammonium sulfate, ā, ka whakakapia te sodium succinate ki te sodium acetate) i roto i tētahi pounamu tīwiri-runga 100 ml (23). I roto i ngā huringa 30-s e rima, i whakatōngia ngā pirepire karāhe 0.1 micron i te ōwehenga rōrahi 1:1 hei wawahi i ngā pūtau, ā, i whakamataohia ki runga i te hukapapa mō ngā meneti 5. I tangohia ngā mea kore e rewa, ngā pūtau kāore i pakaru me ngā pirepire karāhe mā te centrifugation i te 16,000 RCF mō ngā meneti 10 i roto i tētahi microcentrifuge papa. I wehea te kiriuhi i roto i te rotor Ti 70.1 me te 100,000 RCF i roto i te 20 mM tris-HCl (pH 8.0) me te 40/15% (w/w) te pikinga huka mō ngā haora 10.
E ai ki tā mātou mahi o mua, te kimi ārai mate o te tohu His i runga i te PufW (16). I roto i te poto, ko te matatini matua kua purea (11.8 nM) te kiriuhi rānei kei roto te kukū ōrite o te RC (i whakatauhia mā te waikura tango i te whānuitanga rerekētanga kua whakaitihia me te taurite i te kawenga i runga i te tiēre kua pania) i roto i te 2x SDS loading buffer (Merck, UK) i whakawaiwaihia kia rua ngā wā. I wehea ngā pūmua i runga i te tiēre NuPage 12% bis-tris (Thermo Fisher Scientific, UK). I pania he tiēre ki te Coomassie Brilliant Blue (Bio-Rad, UK) hei uta me te tiro i te waeine RC-L. I whakawhitia te pūmua i runga i te tiēre tuarua ki te kiriuhi polyvinylidene fluoride (PVDF) kua whakahohehia e te methanol (Thermo Fisher Scientific, UK) mō te whakamātautau ārai mate. I āraia te kiriuhi PVDF ki roto i te 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v/v) Tween-20 me te 5% (w/v) paura miraka ngako, kātahi ka whakatōkia ki te paturopi tuatahi anti-His (ki roto i te Whakawaiwaihia te ārai paturopi [50 mM tris-HCl (pH 7.6), 150 mM NaCl me te 0.05% (v/v) Tween-20] ki roto i te 1:1000 A190-114A, Bethyl Laboratories, USA) mō ngā haora e 4. I muri i te horoinga e 3 ngā wā mō te 5 meneti i roto i te pūhui paturopi, i whakakotahihia te kiriuhi ki te paturopi tuarua anti-kiore o te horseradish peroxidase (Sigma-Aldrich, UK) (i whakaranua ki te pūhui paturopi 1:10,000). Whakatōkia kia taea ai te kimi (5 meneti i muri i te horoinga e 3 i roto i te pūhui paturopi) mā te whakamahi i te WESTAR ETA C 2.0 chemiluminescence substrate (Cyanagen, Itari) me Amersham Imager 600 (GE Healthcare, UK).
Mā te tuhi i te tohatoha kaha o ia ara ira kua pania, i ia ara whakamātautau ārai mate rānei, me te whakauru i te horahanga i raro i te tihi me te tatau i te ōwehenga kaha o te RC-L (ira kua pania) me te Pūmua-W (immunoassay), i roto i te ImageJ (57) Tukatukahia te ahua. I hurihia ēnei ōwehenga ki ngā ōwehenga molar mā te whakaaro ko te ōwehenga o te RC-L ki te pūmua-W i roto i te tauira RC-LH114-W parakore he 1:1 me te whakataurite i te huinga raraunga katoa kia rite.
Mō ngā rauemi tāpiri mō tēnei tuhinga, tirohia http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
He tuhinga urunga-tuwhera tēnei e tohatohahia ana i raro i ngā tikanga o te Raihana Whakawhiwhinga Auaha. Ka whakaaetia e te tuhinga te whakamahinga, te tohatoha, me te tārua kore here i roto i tētahi momo pāpāho i raro i te whakaritenga me whakahua tika te mahi taketake.
Kia mōhio: Ko tā mātou anake he tuku mai i tō wāhitau īmēra kia mōhio ai te tangata e taunaki ana koe ki te whārangi e hiahia ana koe kia kite rātou i te īmēra, ā, ehara i te pāme. Kāore mātou e hopu i tētahi wāhitau īmēra.
Ka whakamahia tēnei pātai hei whakamātautau mēnā he manuhiri koe, hei ārai hoki i te tuku pāme aunoa.
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
Mā te hanganga taumira teitei o te matatini mahanga mārama 1 i roto i te pokapū tauhohenga e whakarato he māramatanga hou ki ngā whanaketanga 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
Mā te hanganga taumira teitei o te matatini mahanga mārama 1 i roto i te pokapū tauhohenga e whakarato he māramatanga hou ki ngā whanaketanga quinone.
©2021 American Association for the Advancement of Science. mana katoa. Ko AAAS te hoa o HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef me COUNTER. ScienceAdvances ISSN 2375-2548.