I kēia manawa†Wahi noho o kēia manawa: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
ʻO ka hui hoʻouluulu mālamalama kikowaena hana 1 (RC-LH1) ka ʻāpana photosynthetic koʻikoʻi o ka bacteria phototrophic poni. Ua hoʻolauna mākou i ʻelua mau ʻano microscopy cryo-electron o ka hui RC-LH1 mai Rhodopseudomonas palustris. ʻO ka ʻano hoʻonā 2.65-Å o ka hui RC-LH114-W he 14 mau loop subunit LH1 e hoʻopuni ana iā RC, kahi i hoʻopau ʻia e ka protein W, ʻoiai ʻo ka hui me ka ʻole o ka protein-W he RC piha i hoʻopuni ʻia e RC. Pani ʻia he 16 subunit LH1 loop. ʻO ka hoʻohālikelike ʻana o kēia mau ʻano e hāʻawi i ka ʻike i loko o ka dynamics o quinone i loko o ka hui RC-LH1, me nā loli conformational i hoʻoholo ʻole ʻia ma mua i ka wā e hoʻopaʻa ai i ka quinone ma kahi RC QB, a me kahi o nā wahi hoʻopaʻa quinone kōkua, e kōkua ana i ka hoʻoili ʻana iā lākou i RC. ʻO ke ʻano kū hoʻokahi o ka protein W e pale aku i ka pani ʻana o ka loop LH1, no laila e hana ana i kahi kahawai no ka hoʻolalelale ʻana i ka hoʻololi quinone/quinolone.
Hiki i ka ikehu i hāʻawi ʻia e ka photosynthesis ke hoʻomau i ka aneane o nā ola āpau ma ka honua, a he hiki nui kona no ka biotechnology solar. ʻOiai e hoʻolaha ana i ka photosynthesis honua, hōʻike pū nā bacteria phototrophic poni i nā ʻano ikehu like ʻole a me nā hiki metabolic. Hiki iā lākou ke pale aku i ka photosynthesis a ulu ma ke ʻano he bacteria heterotrophic i ka pōʻeleʻele, hiki ke hoʻopaʻa i ka nitrogen a me ke kalapona dioxide, hana i ka hydrogen, a hoʻohaʻahaʻa i nā hui aromatic (1-3). I mea e hāʻawi ai i ka ikehu no kēia mau kaʻina hana, pono e hoʻololi koke ʻia ka mālamalama a me ka maikaʻi i ikehu kemika. Hoʻomaka kēia kaʻina hana i ka wā e omo ai ka complex antenna light-trapping i ka mālamalama a hoʻoili i ka ikehu i hoʻopaʻa ʻia i ke kikowaena hopena (RC), a laila e hoʻomaka ana ka hoʻokaʻawale ʻana i ka uku (4 - 7). ʻO ka ʻāpana kumu o ka photosynthesis i loko o nā bacteria phototrophic poni ua haku ʻia me ke ʻano 2 RC, i hoʻopuni ʻia e ka complex light-harvesting 1 (LH1), e hana ana i ka complex RC-LH1 core. Hoʻokumu ʻia ʻo LH1 e kahi pūʻulu o nā heterodimers αβ curved, kēlā me kēia mea e hoʻopaʻa i ʻelua mau mole chlorophyll bacteria (BChl) a a me hoʻokahi a ʻelua paha carotenoids (8-12). ʻO ka antenna LH1 maʻalahi loa he 16 a i ʻole 17 αβ heterodimers e hoʻopuni ana iā RC (9-13) i loko o kahi loop pani, akā i loko o nā complexes core ʻē aʻe, hoʻopilikia nā peptides transmembrane i ka hoʻomau ʻana o ka LH1 a puni, Ma laila e hoʻolaha ai i ka quinol/quinone diffusion ma waena o RC a me ka cytochrome bc1 complex (11, 13-15). ʻO ka mea kanu poni phototrophic Rhodopseudomonas (Rps.) he mea hoʻohālike hiki ke hoʻomaopopo i ka ikehu a me ka hoʻoili electron e kākoʻo ana i ka photosynthesis. ʻO ke ʻano kristal mua o Rps. ʻO ke kumu hoʻohālike o ka palustris RC-LH1 complex ʻo RC, i hoʻopuni ʻia e 15 heterodimeric LH1 loops, i hoʻopau ʻia e kahi protein ʻike ʻole ʻia i kapa ʻia ʻo "Protein W" (14). Ua ʻike ʻia ʻo Protein-W ma hope ʻo RPA4402, he protein 10.5kDa i hōʻike ʻole ʻia me ʻekolu mau helices transmembrane i wānana ʻia (TMH) (16). Ke hāpai nei mākou e kapa hou i ka inoa o ka rpa4402 gene encoding protein W i pufW e kūlike me ka nomenclature i hoʻohana ʻia no nā genes encoding RC-L, M (pufL, pufM) a me LH1α, β (pufA, pufB) subunits. ʻO ka mea hoihoi, aia wale nō ka protein-W ma kahi o 10% o RC-LH1, e hōʻike ana iā Rps. palustris e hana i ʻelua mau complexes RC-LH1 like ʻole. Maanei, hōʻike mākou i nā ʻano cryo-EM (cryo-EM) kiʻekiʻe o nā complexes koʻikoʻi ʻelua, hoʻokahi me ka protein W a me 14 αβ heterodimers, ʻo kekahi me ka ʻole o ka protein W a me kahi loop 16 Heterodimer LH1 i pani ʻia. Hōʻike kā mākou ʻano i kahi hoʻololi ʻanuʻu i ka hoʻomaopopo ʻana i ka complex RC-LH1 o Rps. palustris, no ka mea, ua kālailai mākou i ka heluna homogeneous o kēlā me kēia ʻano a loaʻa iā mākou ka hoʻonā lawa e hāʻawi maopopo i kēlā me kēia peptide a me nā pigments i hoʻopaʻa ʻia a me nā lipids a me nā quinones pili. ʻO ka hoʻohālikelike ʻana o kēia mau ʻano hana e hōʻike ana ʻo nā protein TMH ʻekolu-W i loaʻa ʻole i loko o kekahi hui RC-LH1 ʻē aʻe a hiki i kēia manawa e hoʻopuka i kahi kahawai quinone e hoʻolalelale i ka hoʻololi quinone/quinolone. Ua ʻike ʻia kekahi mau wahi hoʻopaʻa lipid a me quinone i mālama ʻia, a ua hōʻike mākou i kahi hoʻololi conformational hou ma hope o ka hui pū ʻana o quinone a me RC, kahi mea kūpono paha no ka photosystem II (PSII) RC o nā meaola phototrophic oxygenated. Hāʻawi kā mākou mau ʻike i nā ʻike hou i loko o ke kinetics o ka hoʻopaʻa ʻana a me ka hoʻololi ʻana o quinone/quinolone i loko o ka hui kumu RC-LH1 o nā bacteria phototrophic poni.
I mea e maʻalahi ai ke aʻo kikoʻī ʻana i nā paʻakikī ʻelua i loaʻa ma Rps. palustris, hoʻokaʻawale mākou i kēlā me kēia RC-LH1 ma o nā ʻano biochemical. Ua hoʻomaʻemaʻe ʻia ka paʻakikī protein W-deficient (ma hope aku nei i kapa ʻia ʻo ΔpufW) mai ke ʻano nele i ka gene pufW (16), a hoʻokahi wale nō paʻakikī RC-LH1 hiki ke hana ʻia. Hoʻopuka ʻia ka paʻakikī protein W-containing e kahi ʻano. Hoʻololi ʻia ka protein W o kēia ʻano me kahi tag 10x His ma kona C-terminus, i hiki ke hoʻohui maikaʻi ʻia ka paʻakikī protein W-containing me ka hapa nui o ka protein W nele ma ka hoʻopaʻa ʻana i ka metala. Hoʻokaʻawale maikaʻi ʻia ka paʻakikī (16) Affinity Chromatography (IMAC).
E like me ka mea i hōʻike ʻia ma ke Kiʻi 1, aia i loko o nā hui ʻelua kahi ʻāpana liʻiliʻi ʻekolu RC (RC-L, RC-M a me RC-H) i hoʻopuni ʻia e kahi antenna LH1. ʻO ke ʻano 2.80-A o ka hui nele i ka protein-W e hōʻike ana i 16 αβ heterodimers, e hana ana i kahi loop LH1 pani e hoʻopuni piha ana iā RC, i kapa ʻia ma hope aku ʻo ka hui RC-LH116. ʻO ke ʻano 2.65Å o ka hui i loaʻa ka protein-W he 14-heterodimer LH1 i hoʻopau ʻia e ka protein-W, i kapa ʻia ma hope aku ʻo RC-LH114-W.
(A a me B) Hōʻike ʻana o ka ʻili o ka hui. (C a me D) Nā pigments i hoʻopaʻa ʻia i hōʻike ʻia i loko o nā koʻokoʻo. (E a ​​me F) ʻO nā complexes i ʻike ʻia mai ka ʻili cytoplasmic he mau peptides a me nā subunits LH1 i hōʻike ʻia i loko o nā kiʻi ʻoniʻoni, a ua helu ʻia ma ka ʻaoʻao o ka uaki mai ka protein-W gap [kūlike me ka helu ʻana o Rba. sphaeroides complex (13)]. No LH1-α, he melemele ke kala o ka subunit protein; no LH1-β, he uliuli ke kala o ka subunit protein; no protein-W, he ʻulaʻula ka protein; no RC-H, he cyan; no RC-L, he ʻAlani; no RC-M, magenta. Hōʻike ʻia nā Cofactors e nā koʻokoʻo, hōʻike ka ʻōmaʻomaʻo i nā molekala BChl a me BPh a, hōʻike ka poni i nā carotenoids, a hōʻike ka melemele i nā molekala UQ10. (G a me H) ʻIke nui ʻia o ka protein-W gap ma ka ʻāpana like o ka complex RC-LH114-W (G) a me ka complex RC-LH116 (H). Hōʻike ʻia nā Cofactors ma ke ʻano o ka hoʻopiha hakahaka, hōʻike ʻia ka quinone chelated ma ka uliuli. Hōʻike ʻia ka hakahaka protein-W e kahi laina polū i kahakaha ʻia ma (G), a ʻo nā lua liʻiliʻi kahi e hoʻolaha ai ka quinone/quinolol ma ke apo LH116 ua hōʻike ʻia e kahi laina ʻeleʻele i kahakaha ʻia ma (H).
Hōʻike ka Kiʻi 1 (A a me B) i ka RC i hoʻopuni ʻia e nā ʻāpana hāmama a pani ʻia paha o nā heterodimers LH1αβ, kēlā me kēia e hoʻopaʻa i ʻelua BChl a me hoʻokahi carotenoid (Kiʻi 1, C a me D). Ua hōʻike nā haʻawina mua ʻo Rps ka paʻakikī LH1. Ma ke ala biosynthetic o spirulina xanthin, loaʻa i kēia mau ʻano nā heluna hui o nā carotenoids (17). Eia nō naʻe, ʻo ka spiropyrroxanthin ka carotenoid dominant a ua kūpono kona density. No laila, ua koho mākou e hoʻohālike i ka spiroxanthin ma nā wahi hoʻopaʻa LH1 āpau. ʻO nā polypeptides alpha a me beta he mau TMH hoʻokahi me nā ʻāpana waho membrane pōkole (Kiʻi 1, A, B, E, a me F). ʻOiai ʻaʻole i ʻike ʻia ka density o 17 mau koena ma ka C-terminus, ua ʻoki ʻia ka alpha polypeptide mai Met1 a i Ala46 ma nā paʻakikī ʻelua. Ua hoʻemi ʻia ka β polypeptide mai Gly4 a i Tyr52 ma RC-LH116, a mai Ser5 a i Tyr52 ma RC-LH114-W. ʻAʻohe ʻike ʻia ka nui o 3 a i ʻole 4 N-terminal a i ʻole 13 C-terminal residues (Kiʻi S1). Ua hōʻike ka loiloi spectrometry mass o ka hui RC-LH1 i hoʻomākaukau ʻia mai ke ʻano hihiu ʻo ka ʻāpana nalowale ka hopena o ka ʻoki heterologous o kēia mau peptides (Kiʻi S1 a me S2). Ua ʻike pū ʻia ka formylation N-terminal o α-Met1 (f). Ua hōʻike ka loiloi he α-peptide nā koena fMet1 a i Asp42/Ala46/Ala47/Ala50, a ʻo ka β-peptide nā koena Ser2 a i Ala53, kahi e kūlike maikaʻi me ka palapala ʻāina density EM haʻahaʻa.
ʻO ka hoʻonohonoho ʻana o α-His29 a me β-His36 e hoʻohālikelike i nā BChls; hui pū kēlā me kēia αβ heterodimer me kona mau hoalauna e hana i kahi loop hāmama (RC-LH114-W) a i ʻole kahi loop pani (RC-LH116) a puni ka RC ʻO ka exciton coupled pigment array (Kiʻi 1, C a me D). Ke hoʻohālikelike ʻia me ka hui 877 nm o RC-LH114-W, ʻo ka 880 nm absorption red shift o RC-LH116 he 3 nm (Kiʻi 2A). Eia nō naʻe, ua aneane like ka circular dichroism spectrum (Kiʻi 2B), e hōʻike ana ʻoiai he ʻokoʻa maopopo ma waena o nā loop hāmama a pani ʻia, ua like loa ke kaiapuni kūloko o BChls. ʻO ka absorption redshift paha ka hopena o ka emi ʻana o ka neʻe ʻana o ka thermal a me ka hoʻonui ʻana i ke kūpaʻa ma ka loop pani (18, 19), ka loli o ka pigment coupling i hoʻokumu ʻia e ka loop pani (20, 21), a i ʻole ka hui pū ʻana o kēia mau hopena ʻelua (11).
(A) ʻO ka spectrum absorption ultraviolet/ʻike ʻia/kokoke-infrared, nā piko o ia mea i hōʻailona ʻia me kā lākou pigments like a hoʻomaʻamaʻa ʻia i ka piko BPh ma 775 nm. (B) ʻO ka spectrum dichroism circular i hoʻomaʻamaʻa ʻia i ka absorbance BChl ma 805 nm. (C a me D) Nā spectra ΔA i koho ʻia mai nā spectra absorption i hoʻoholo ʻia i ka manawa o ka paʻakikī RC-LH114-W (C) a me ka paʻakikī RC-LH116 (D). No ka hoʻohālikelike maikaʻi ʻana, ua hoʻomaʻamaʻa ʻia nā spectra āpau i ∆A o −A ma 0.2 ps. (E) ʻO ka wikiwiki o ka oxidation cytochrome c2 ma hope o ka irradiation i ke alo o nā ʻano like ʻole o UQ2 (e nānā i ke Kiʻi S8 no ka ʻikepili maka). (F) I loko o nā cell i ulu ʻia ma lalo o ke kukui haʻahaʻa, waena a kiʻekiʻe paha (10, 30 a i ʻole 300μMm-2 s-1, kēlā me kēia), ʻo ka protein W a me nā subunits RC-L i loko o ka paʻakikī i hoʻomaʻemaʻe ʻia a me ka lakio membrane i hoʻokaʻawale ʻia. E hoʻoholo i ka pae protein ma o ka SDS-polyacrylamide gel electrophoresis a me ka immunoassay (e nānā i ke Kiʻi S9 no ka ʻikepili maka). E hoʻoholo i ka lakio e pili ana i ka hui RC-LH114-W i hoʻomaʻemaʻe ʻia. ʻO ka lakio stoichiometric o RC-L i ka protein-W o ka hui he 1:1.
ʻOi aku ka kokoke o nā BChl ma ke kūlana 1 i loko o ka loop αβ14 deformed o RC-LH114-W (Kiʻi 1, A, C, a me E) i ka mea hāʻawi mua RC (P) ma 6.8Å ma mua o nā BChl like ma RC-LH116 (Kiʻi 1, B, D, a me F, a me ke Kiʻi S3); eia naʻe, hōʻike nā kinetics absorption transient o nā complexes ʻelua no RC-LH114-W a me RC-LH116, ʻo nā constants manawa hoʻoili ikehu excitation mai LH1 a i RC he 40 ±4 a me 44 ±3 ps (Kiʻi 2). , C a me D, Kiʻi S4 a me ka Papa S2). ʻAʻohe ʻokoʻa koʻikoʻi i ka hoʻoili uila i loko o RC (Kiʻi S5 a me ka kikokikona hoʻohui pili). Manaʻo mākou ʻo ka pili kokoke o ka manawa hoʻoili ikehu ma waena o LH1 a me RC-P ma muli o ka mamao like, ke kihi a me ka ikehu hiki o ka hapa nui o BChl i nā loops LH1 ʻelua. Me he mea lā ʻaʻole wikiwiki ka ʻimi ʻana i ke ʻano ikehu LH1 e hiki ai i ka mamao liʻiliʻi loa ma mua o ka hoʻoili ikehu pololei mai nā wahi suboptimal a i RC. Hiki i ka loop LH1 open-loop ma RC-LH114-W ke hana i kahi neʻe thermal liʻiliʻi ma lalo o nā kūlana mahana haʻahaʻa no ka nānā ʻana i ke ʻano, a aia kahi conformation apo αβ14 lōʻihi ma ka mahana o ka lumi mai ka mamao pigmentation o βBChls ma ke kūlana o RC 1.
Aia i loko o ka hui RC-LH116 he 32 BChls a me 16 carotenoids, a ʻo kona hoʻonohonoho holoʻokoʻa ua like ia me ka mea i loaʻa mai Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 strain (PDB ID 7C9R) (12) a me ka limu ʻōmaʻomaʻo (Blc.viridis) (PDB ID 6ET5) (10). Ma hope o ke kaulike ʻana, ua ʻike ʻia nā ʻokoʻa liʻiliʻi wale nō ma nā kūlana o nā αβ heterodimers, ʻoiai ʻo 1-5, 15, a me 16 (Kiʻi S6). He hopena koʻikoʻi ka loaʻa ʻana o ka protein-W i ke ʻano o LH1. Hoʻopili ʻia kāna mau TMH ʻekolu e nā loop pōkole, me ka N-terminal ma ka ʻaoʻao lumen o ka hui a me ka C-terminal ma ka ʻaoʻao cytoplasmic (Nā Kiʻi 1A a me 3, A a D). He hydrophobic nui ʻo Protein-W (Kiʻi 3B), a launa pū ʻo TMH2 lāua ʻo TMH3 me LH1αβ-14 e hana i kahi ʻili transmembrane (Kiʻi 3, B a me E a i G). ʻO ka interface ka mea nui i haku ʻia me nā koena Phe, Leu a me Val ma ka ʻāpana transmembrane. Hoʻopili ʻia kēia mau koena me nā waikawa amino hydrophobic a me nā pigments αβ-14. Hāʻawi pū kekahi mau koena polar i ka launa pū ʻana, me ka pilina hydrogen ma waena o W-Thr68 a me β-Trp42 ma ka ʻili o ka lua paʻakikī (Kiʻi 3, F a me G). Ma ka ʻili o ka cytoplasm, pili ʻo Gln34 i ka hui keto o nā carotenoids αβ-14. Eia kekahi, ua hoʻonā ʻia ka molekala n-dodecyl β-d-maltoside (β-DDM), a ua hoʻolōʻihi ʻia kona huelo hydrophobic i ka interface ma waena o ka protein-W a me αβ-14, a aia paha ka huelo lipid i loko o ke kino. Ua ʻike pū mākou ua kokoke loa nā wahi hoʻonā C-terminal o ka protein W a me RCH, akā ʻaʻole i loko o ka laulā o ka hoʻokumu ʻana i nā pilina kikoʻī (Kiʻi 1, A a me E). Eia nō naʻe, aia paha nā pilina ma nā waikawa amino C-terminal i hoʻoholo ʻole ʻia o kēia mau protein ʻelua, kahi e hāʻawi ai i kahi ʻano hana no ka hoʻolimalima ʻana o ka protein-W i ka wā o ka ʻākoakoa ʻana o ka paʻakikī RC-LH114-W.
(A) ʻO Protein-W, kahi e kū pono ana i ka pilina me LH1αβ14 ma ke ʻano kiʻiʻoniʻoni, he kaulahao ʻaoʻao ʻano koʻokoʻo (ʻulaʻula), i hōʻike ʻia ma kahi ʻāpana o ke kiʻikuhi hiki electrostatic (ʻili hina moakaka me ka pae contour o 0.13). (B) Hōʻike ʻia ʻo Protein-W e kahi ʻili kala hydrophobic. Hōʻike ʻia nā wahi polar a me nā wahi i hoʻopiʻi ʻia ma ke cyan, hōʻike ʻia nā wahi hydrophobic i ke keʻokeʻo, a hōʻike ʻia nā wahi hydrophobic ikaika i ka ʻalani. (C a me D) Hōʻike ʻia ʻo Protein-W ma ke kiʻiʻoniʻoni, ua like kona kuhikuhi ʻana me (A) (C), a hoʻohuli ʻia e 180° (D). Wahi a ke kūlana ma ke kaʻina, lawe nā koena i hiki ke hoʻokaʻawale ʻia i kahi ʻano kala ānuenue, kahi i uliuli ai ka N-terminal a ʻulaʻula ka C-terminal. (E) ʻO Protein-W ma ka hiʻohiʻona like me (A), a ʻo nā koena ma ka pilina o protein-W:LH1 i hōʻike ʻia e nā koʻokoʻo me nā māka i hoʻopili ʻia. (F) Ua hoʻohuli ʻia ʻo Protein-W he 90° e pili ana iā (E) a me LH1αβ14 ma ke kiʻi ʻoniʻoni, a e pili ana i nā koena interface ma ke kiʻi ʻāpana. Ua lepili ʻia nā koena e kau ana mai ka beta polypeptide. Ua hōʻike ʻia ka cofactor ma ke ʻano he ʻāpana e kūlike ana i ke kala o ke Kiʻi 1, ua hōʻike ʻia ka β-DDM i hoʻokahe ʻia ma ke hina hina, a ua hōʻike ʻia ka oxygen ma ka ʻulaʻula. (G) Ua hoʻohuli ʻia ka nānā ʻana ma (F) he 180°, me nā koena koʻikoʻi o ka alpha polypeptide i lepili ʻia.
Hoʻololi ʻo Protein-W i kahi αβ heterodimer (ka 15 ma ke Kiʻi 1F), no laila e pale ana i ka pani ʻana o ka loop a me ka hoʻohuli ʻana i nā αβ heterodimers mua ʻekolu. Ua ʻike ʻia ʻo ke kihi inclination kiʻekiʻe loa o ka αβ-1 heterodimer mua e pili ana i ke kiʻiʻoniʻoni maʻamau he 25° a 29° (Kiʻi 1, A a me E), i hoʻokumu ʻia e ka inclination 2° a 8° o αβ-1 ma RC A sharp contrast-LH116 (Kiʻi 1, B a me F). Hoʻohuli ʻia nā heterodimers ʻelua a me ke kolu ma 12° a 22° a me 5° a 10°, kēlā me kēia. Ma muli o ke keakea steric o RC, ʻaʻole i hoʻokomo ka tilt o αβ-1 i ka lua o ka hui o αβ (e kūlike ana me ka 16th αβ ma ke Kiʻi 1F), no laila e hana ana i kahi hakahaka maopopo i loko o ke apo LH1 (Kiʻi 1, A a me E). Ma muli o ka nele o ʻelua αβ heterodimers, me ka nalowale ʻana o ʻehā BChl a me ʻelua carotenoids, ʻaʻohe o nā carotenoids e hoʻopaʻa i ka subunit αβ-1 i wili ʻia, e hopena ana i kahi apo LH114-W e loaʻa ana he 13 carotenoids Vegetarian a me 28 BChls. ʻOi aku ka haʻahaʻa o nā kuhi hoʻonā kūloko o nā complexes ʻelua ma nā ʻāpana αβ1 a 7 ma mua o ke koena o ka loop LH1, kahi e hōʻike ai i ka plasticity kūlohelohe o ka subunit LH1 e pili ana i ka pūnaewele RC QB (Kiʻi 4).
Ua hōʻike ʻia nā kiʻi o RC-LH114-W (A a me B) a me RC-LH116 (C a me D) mai ka ʻike luna/ʻike ʻaoʻao like (A a me B) (A a me C) a me ka ʻili lua o ke Kiʻi 1. (B a me D). Ua hōʻike ʻia nā kī kala ma ka ʻākau.
ʻO ka hui kumu ʻē aʻe me ka lakio stoichiometric o 1:14 ʻo ia ka Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Eia nō naʻe, ʻaʻohe homology maopopo o ka protein W a me PufX, a he hopena koʻikoʻi ko lākou i kā lākou mau ʻano LH1. ʻO PufX kahi TMH hoʻokahi me kahi kikowaena cytoplasmic N-terminal e launa pū me ka ʻaoʻao cytoplasmic o ka subunit RC-H (13) ma kahi kūlana e pili ana iā Rps. palustris LH116αβ-16. Hoʻokumu ʻo PufX i kahi kahawai no ka hoʻololi quinone/quinolone ma waena o RC-LH1 a me ka hui cytochrome bcl a aia i loko o nā hui kumu Rba āpau. sphaeroides (13). ʻOiai aia ka interface monomer-monomer ma Rba. Aia ka dimer sphaeroides RC-LH1-PufX ma ke kūlana hoʻopaʻa o ka protein W ma RC-LH114-W, a ʻo ka hakahaka i hoʻokumu ʻia e PufX a me ka protein-W aia ma kahi kūlana like (Kiʻi S7A). Ua kūlike pū ka hakahaka ma RC-LH114-W me ke kahawai quinone hypothetical (8) o Pseudomonas rosea LH1, i hoʻokumu ʻia e nā peptides ʻaʻole pili i ka protein W a i ʻole PufX (Kiʻi S7B). Eia kekahi, ʻo ke kahawai quinone ma Blc. Aia ka ʻōmaʻomaʻo emerald LH1 i hoʻokumu ʻia ma ka hoʻokaʻawale ʻana i hoʻokahi subunit γ (7) ma kahi kūlana like (Kiʻi S7C). ʻOiai ua hoʻopili ʻia e nā protein like ʻole, ʻo ke ʻano o kēia mau kahawai quinone/quinolol ma kahi kūlana maʻamau i ka hui RC-LH1 me he mea lā he laʻana o ka ulu ʻana o ka hui ʻana, e hōʻike ana e hiki i ka hakahaka i hana ʻia e ka protein W ke hana ma ke ʻano he kahawai quinone.
ʻO ka hakahaka ma ka loop LH114-W e ʻae i ka hoʻokumu ʻana o kahi ʻāpana membrane hoʻomau ma waena o ka hakahaka o loko o ka complex RC-LH114-W a me ka membrane nui (Kiʻi 1G), ma mua o ka hoʻopili ʻana i nā kikowaena ʻelua ma o kahi pore protein e like me nā protein. Ua like ka complex RC-LH116 me kahi Tch pani. ʻO ka complex needle-like (22) (Kiʻi 1H). ʻOiai ʻoi aku ka wikiwiki o ka diffusion o ka quinone ma o ka membrane ma mua o ka diffusion ma o ke kahawai protein haiki, hiki i ka loop LH114-W hāmama ke ʻae i ka RC turnover wikiwiki ma mua o ka loop LH116 pani, a ʻoi aku ka palena o ka diffusion o ka quinone i loko o ka RC. I mea e hoʻāʻo ai inā pili ka protein W i ka hoʻololi ʻana o nā quinones ma o RC, ua hana mākou i kahi cytochrome oxidation assay ma kahi ʻano o ubiquinone 2 (UQ2) (kahi analogue o UQ10 kūlohelohe me kahi huelo isoprene pōkole) (Kiʻi 2E). ʻOiai ke keakea nei ka loaʻa ʻana o ka chelated quinone i ka hoʻoholo pololei ʻana o ka Michaelis constant (ʻo RC-LH114-W a me RC-LH116 he kūpono no 0.2±0.1μM a me 0.5±0.2μM, kēlā me kēia), ʻo ka nui loa o RC-LH114-W (4.6±0.2 e-RC-1 s-1) he 28±5% ʻoi aku ka nui ma mua o RC-LH116 (3.6±0.1 e-RC-1 s-1).
Ua manaʻo mua mākou aia ka protein-W ma kahi o 10% o ka paʻakikī koʻikoʻi (16); maanei, ʻo ka nui o ka noho ʻana o nā cell ulu haʻahaʻa, waena, a me ke kukui kiʻekiʻe he 15 ± 0.6%, 11 ± 1% a me 0.9 ± 0.5, kēlā me kēia % (Kiʻi 2F). Ua hōʻike ka hoʻohālikelike quantitative o ka mass spectrometry ʻaʻole i hōʻemi ka hoʻohui ʻana o ka histidine tag i ka nui o ka protein-W i hoʻohālikelike ʻia me nā ʻano wild-type (P = 0.59), no laila ʻaʻole kēia mau pae he artifact o ka protein-W i hoʻololi ʻia (Kiʻi S10). Eia naʻe, ʻo kēia noho haʻahaʻa o ka protein-W i loko o ka paʻakikī RC-LH1 e ʻae i kekahi mau RC e hoʻololi i ka wikiwiki, no laila e hōʻemi ana i ka hoʻololi lohi o ka quinone/quinolone i loko o ka paʻakikī RC-LH116. Ua ʻike mākou ʻaʻole kūlike ka nui o ka noho kukui kiʻekiʻe me ka ʻikepili transcriptomics hou, e hōʻike ana e hoʻonui ka hōʻike ʻana o ka gene pufW ma lalo o ka mālamalama ikaika (Kiʻi S11) (23). He huikau ka ʻokoʻa ma waena o ka palapala pufW a me ka hoʻokomo ʻia ʻana o ka protein-W i loko o ka hui RC-LH1 a hiki ke hōʻike i ka hoʻoponopono paʻakikī o ka protein.
Ma RC-LH114-W, ua hoʻokaʻawale ʻia a hoʻohālike ʻia he 6 cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) a me 29 mau molekala β-DDM i loko ona he 6 CDL, 24 POPC, 2 POPG a me 12 βDDM. RC-LH116 (Kiʻi 5, A a me B). Ma kēia mau ʻano ʻelua, ua kokoke ka CDL ma ka ʻaoʻao cytoplasmic o ka complex, ʻoiai ʻo POPC, POPG a me β-DDM aia ka hapa nui ma ka ʻaoʻao luminal. Ua hoʻokaʻawale ʻia ʻelua mau molekala lipid a me nā molekala detergent ma ka ʻāpana αβ-1 a i αβ-6 o ka complex RC-LH114-W (Kiʻi 5A), a ua hoʻokaʻawale ʻia ʻelima ma ka ʻāpana like o RC-LH116 (Kiʻi 5B). Ua loaʻa nā lipid hou aʻe ma kēlā ʻaoʻao o ka paʻakikī, ʻo CDL hoʻi, i hōʻiliʻili ʻia ma waena o RC a me αβ-7 a i αβ-13 (Kiʻi 5, A a me B). Aia nā lipid a me nā detergents ʻē aʻe i hoʻoponopono ʻia ma ke ʻano ma waho o ke apo LH1, a ʻo nā kaulahao acyl i hoʻonā maikaʻi ʻia e hoʻolōʻihi ma waena o nā subunits LH1, i kapa ʻia he β-DDM ma RC-LH114-W, a ua wehewehe ʻia ʻo β-DDM ma RC A hui pū ʻia o β-DDM a me POPC-LH116. ʻO nā kūlana like o nā lipids chelating a me nā detergents i loko o kā mākou ʻano e hōʻike ana he mau wahi hoʻopaʻa pili kino lākou (Kiʻi S12A). Loaʻa pū kekahi kūlike maikaʻi i nā kūlana o nā molekala like ma Tch. ʻO Gentle a me Trv. ʻO ke ʻano 970 RC-LH1s (Kiʻi S12, B a i E) (9, 12) a me nā koena hoʻopaʻa hydrogen o ka hui poʻo lipid i hōʻike i ka mālama maikaʻi ʻana i ka hoʻonohonoho ʻana o ke kaʻina (Kiʻi S13), e hōʻike ana i ka CDL i mālama ʻia e hoʻopaʻa iā RC (24), hiki ke mālama ʻia kēia mau wahi i loko o ka paʻakikī RC-LH1.
(A a me B) Hōʻike ʻia nā peptides RC-LH114-W (A) a me RC-LH116 (B) e nā kiʻi ʻoniʻoni, a hōʻike ʻia nā pigments e nā koʻokoʻo, me ka hoʻohana ʻana i ke ʻano kala ma ke Kiʻi 1. Hōʻike ʻia nā lipids i ka ʻulaʻula, a hōʻike ʻia nā detergents i ka hina hina. ʻO UQ i hoʻopaʻa ʻia i nā wahi RC QA a me QB he melemele, ʻoiai ʻo UQ i hoʻokaʻawale ʻia he uliuli. (C a me D) ʻO nā hiʻohiʻona like me (A) a me (B), me nā lipids i haʻalele ʻia. (E a ​​i G) ʻIke nui ʻia o Q1(E), Q2(F) a me Q3(G) mai RC-LH116, me nā kaulahao ʻaoʻao e hoʻohuli kekahi i kekahi. Hōʻike ʻia nā pilina hydrogen ma ke ʻano he mau laina ʻeleʻele i kahakaha ʻia.
Ma RC-LH116, ua hoʻokaʻawale ʻia ʻo RC QA a me QB UQ, ka mea e komo ana i ka hoʻoili uila i ke kaʻina hana hoʻokaʻawale uku, i loko o ko lākou mau wahi hoʻopaʻa. Eia nō naʻe, ma RC-LH114-W, ʻaʻole i hoʻonā ʻia ka QB quinone a e kūkākūkā kikoʻī ʻia ma lalo nei. Ma waho aʻe o nā quinones QA a me QB, ua hoʻokaʻawale ʻia ʻelua mau molekala UQ chelated (aia ma waena o nā apo RC a me LH1) i loko o ka ʻano RC-LH114-W e like me kā lākou mau hui poʻo i hoʻonā maikaʻi ʻia (aia ma Q1 a me Q2, kēlā me kēia). hakahaka). Kiʻi 5C). Ua hāʻawi ʻia ʻelua mau ʻāpana isoprene i Q1, a ua hoʻonā ka palapala ʻāina density i nā huelo isoprene 10 piha o Q2. Ma ke ʻano o RC-LH116, ua hoʻonā ʻia ʻekolu mau molekala UQ10 chelated (Q1 a i Q3, Kiʻi 5D), a he maopopo ka density o nā molekala a pau ma loko o ka huelo (Kiʻi 5, D a i G). Ma nā ʻano ʻelua, he kūlike maikaʻi loa nā kūlana o nā hui poʻo quinone o Q1 a me Q2 (Kiʻi S12F), a launa pū lākou me RC wale nō. Aia ʻo Q1 ma ke komo ʻana o ka hakahaka W o RC-LH114-W (Kiʻi 1G a me 5, C, D a me E), a aia ʻo Q2 ma kahi kokoke i kahi hoʻopaʻa QB (Kiʻi 5, C, D) a me F). ʻO nā koena L-Trp143 a me L-Trp269 i mālama ʻia ua kokoke loa i Q1 a me Q2 a hāʻawi i nā pilina π-stacking hiki (Kiʻi 5, E a me F, a me Kiʻi S12). ʻO L-Gln88, 3.0 Å mai ka oxygen distal o Q1, hāʻawi i kahi pilina hydrogen ikaika (Kiʻi 5E); mālama ʻia kēia koena ma nā RC āpau koe wale nō ka pilina mamao loa (Kiʻi S13). Ua pani ʻia ʻo L-Ser91 no Thr ma ka hapa nui o nā RC ʻē aʻe (Kiʻi S13), he 3.8 Angstroms mai ka methyl oxygen o Q1, a hiki ke hāʻawi i nā pilina hydrogen nāwaliwali (Kiʻi 5E). ʻAʻole ʻike ʻia he pilina kikoʻī ko Q3, akā aia ma ka ʻāpana hydrophobic ma waena o ka subunit RC-M a me ka subunit LH1-α 5 a 6 (Kiʻi 5, D a me G). Ua hoʻoholo ʻia hoʻi ʻo Q1, Q2 a me Q3 a i ʻole nā ​​​​quinones chelated kokoke ma Tch. Gentle, Trv. Strain 970 a me Blc. Kuhikuhi ke ʻano iris (9, 10, 12) i kahi pūnaewele hoʻopaʻa quinone kōkua i mālama ʻia ma ka paʻakikī RC-LH1 (Kiʻi S12G). ʻO nā UQ ʻelima i hoʻopau ʻia ma RC-LH116 e kūlike maikaʻi me ka 5.8 ± 0.7 o kēlā me kēia paʻakikī i hoʻoholo ʻia e ka chromatography wai hana kiʻekiʻe (HPLC), ʻoiai ʻo nā UQ ʻekolu i hoʻopau ʻia ma RC-LH114-W ua haʻahaʻa ma mua o ka ʻO ka waiwai i ana ʻia o 6.2 ± 0.3 (Kiʻi S14) e hōʻike ana aia nā molekala UQ i hoʻoholo ʻole ʻia i loko o ka hale.
Loaʻa i nā polypeptides L a me M pseudo-symmetric ʻelima mau TMH a hana i kahi heterodimer e hoʻohui i hoʻokahi dimer BChl, ʻelua monomers BChl, ʻelua monomers bacteriophage (BPh), a me hoʻokahi hao non-Heme a me hoʻokahi a ʻelua mau molekala UQ10. Ma o ke alo o nā pilina hydrogen ma ka hui ketone terminal a me kona hōʻiliʻili ʻana i ʻike ʻia ma Rps, ua hoʻokomo ʻia nā carotenoids i loko o ka M-subunit, i kapa ʻia ʻo cis-3,4-dehydroorhodopin. ʻAno (25). Hoʻopaʻa ʻia ka ʻāpana membrane waho o RC-H i ka membrane e kahi TMH hoʻokahi. Ua like ke ʻano RC holoʻokoʻa me nā subunit ʻekolu RC o nā ʻano pili (e like me Rba). sphaeroides (PDB ID: 3I4D). ʻO nā macrocycles o BChl a me BPh, ka iwi kuamoʻo carotenoid a me ka hao non-heme e uhi ana i loko o ka pae hoʻonā o kēia mau ʻano, e like me ka hui poʻo UQ10 ma kahi QA a me ka quinone QB ma RC-LH116 (Kiʻi S15).
ʻO ka loaʻa ʻana o ʻelua mau ʻano RC me nā helu noho ʻana o ka pūnaewele QB like ʻole e hāʻawi i kahi manawa hou e nānā ai i nā loli conformational kūlike e hele pū ana me ka hoʻopaʻa ʻana o QB quinone. I loko o ka paʻakikī RC-LH116, aia ka QB quinone ma ke kūlana "proximal" i hoʻopaʻa piha ʻia (26), akā ʻo ka hoʻokaʻawale ʻana o RC-LH114-W ʻaʻohe QB quinone. ʻAʻohe QB quinone ma RC-LH114-W, he mea kupanaha ia no ka mea he hana ka paʻakikī, ʻoi aku ma mua o ka paʻakikī RC-LH116 me ka QB quinone i hoʻonā ʻia ma ke ʻano. ʻOiai ʻo nā apo LH1 ʻelua e chelate ma kahi o ʻeono quinones, ʻelima i hoʻonā ʻia ma ke ʻano i loko o ke apo RC-LH116 pani ʻia, ʻoiai ʻekolu wale nō i kaupalena ʻia ma ke ʻano i loko o ke apo RC-LH114-W hāmama. Hiki i kēia maʻi hoʻonui ke hōʻike i ka wikiwiki o ka hoʻololi ʻana o nā pūnaewele RC-LH114-W QB, nā kinetics quinone wikiwiki i loko o ka paʻakikī, a me ka hoʻonui ʻana i ka hiki ke hele i ka loop LH1. Manaʻo mākou ʻo ka nele o ka UQ ma kahi RC QB o RC-LH114-W he hopena paha ia o kahi paʻakikī a ʻoi aku ka ikaika, a ua hoʻopaʻa koke ʻia kahi QB o RC-LH114-W i ka hoʻololi UQ. Hōʻike ke kahua kikoʻī (ua pani ʻia ke komo ʻana i kahi QB) i ke ʻano o kēia hana.
Me ka ʻole o QB, ʻo ka hoʻohuli ʻana o L-Phe217 i kahi kūlana i kūlike ʻole me ka hoʻopaʻa ʻana o UQ10, no ka mea, e hoʻoulu ia i kahi collision spatial me ka ʻāpana isoprene mua o ka huelo (Kiʻi 6A). Eia kekahi, ʻike maopopo ʻia nā loli conformational nui, ʻoiai ka helix de (helix pōkole ma ka loop ma waena o TMH D a me E) kahi i hoʻoneʻe ʻia ai ʻo L-Phe217 i ka ʻeke hoʻopaʻa QB a me ka hoʻohuli ʻana o L-Tyr223 (Kiʻi 6A) No ka uhaki ʻana i ka pilina hydrogen me ka ʻōnaehana M-Asp45 a pani i ke komo ʻana o ka pūnaewele hoʻopaʻa QB (Kiʻi 6B). Ke pivots nei ʻo Helix de ma kona kumu, ua hoʻoneʻe ʻia ka Cα o L-Ser209 e 0.33Å, ʻoiai ua hoʻoneʻe ʻia ka L-Val221Cα e 3.52Å. ʻAʻohe loli i ʻike ʻia ma TMH D a me E, hiki ke hoʻohui ʻia ma nā ʻano ʻelua (Kiʻi 6A). E like me kā mākou ʻike, ʻo kēia ka ʻano mua ma ka RC kūlohelohe e pani ana i ka pūnaewele QB. ʻO ka hoʻohālikelike ʻana me ke ʻano piha (QB-bound) e hōʻike ana ma mua o ka hoʻemi ʻia ʻana o ka quinone, pono kahi hoʻololi conformational e hoʻokomo iā ia i loko o ka quinone. Hoʻohuli ʻo L-Phe217 e hana i kahi pilina π-stacking me ka hui poʻo quinone, a neʻe ka helix i waho, e ʻae ana i ka iwi o L-Gly222 a me ke kaulahao ʻaoʻao o L-Tyr223 e hana i kahi pūnaewele hoʻopaʻa hydrogen me kahi ʻano hoʻopaʻa hydrogen paʻa (Kiʻi 6, A a me C).
(A) Kiʻi ʻoniʻoni e pili ana o ka hologram (kaula L, kaulahao ʻalani/M, magenta) a me ka apo (hina) ʻano, kahi i hōʻike ʻia ai nā koena koʻikoʻi ma ke ʻano o kahi hōʻike e like me ke koʻokoʻo. Hōʻike ʻia ʻo UQ10 e kahi kaha melemele. Hōʻike ka laina kiko i nā pilina hydrogen i hoʻokumu ʻia i loko o ka ʻano holoʻokoʻa. (B a me C) ʻO ka hōʻike ʻana o ka ʻili o ka apolipoprotein a me ke ʻano apo holoʻokoʻa, e hōʻike ana i ka oxygen kaulahao ʻaoʻao o L-Phe217 i ka uliuli a me L-Tyr223 i ka ʻulaʻula, kēlā me kēia. He ʻalani ka subunit L; ʻaʻole i kala ʻia nā subunit M a me H. (D a me E) Apolipoprotein (D) a me ka holoʻokoʻa (E) nā pūnaewele RC QB [kala e (A) kēlā me kēia] a me Thermophilus thermophilus PSII (ʻōmaʻomaʻo, uliuli me ka quinone plastic; PDB ID: 3WU2) Hoʻohālikelike (58).
ʻOiai ua manaʻo ʻole ʻia, ʻoiai ua loaʻa kekahi mau ʻano o nā RC QB-deficient me ka ʻole o LH1, ʻaʻole i hōʻike mua ʻia nā loli conformational i ʻike ʻia ma kēia haʻawina. ʻO kēia mau mea e komo pū me ke ʻano hoʻopau QB mai Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) a me Rba. sphaeroides (PDB ID: 1OGV) (29), ʻo ia mau mea a pau ua aneane like me ko lākou ʻano QB holoʻokoʻa. Ua hōʻike ka nānā pono ʻana o 3PRC ua hoʻopaʻa nā molekala detergent LDAO (Lauryl Dimethyl Amine Oxide) ma ke komo ʻana o ke kūlana QB, kahi e pale ai i ka hoʻonohonoho hou ʻana i loko o kahi conformation pani. ʻOiai ʻaʻole e palaho ka LDAO ma ke kūlana like ma 1EYS a i ʻole 1OGV, ua hoʻomākaukau ʻia kēia mau RC me ka hoʻohana ʻana i ka detergent like a no laila hiki ke hana i ka hopena like. ʻO ke ʻano kristal o Rba. ʻO Sphaeroides RC i hoʻopili ʻia me ka cytochrome c2 (PDB ID: 1L9B) me he mea lā he wahi QB pani ʻia. Eia nō naʻe, i kēia hihia, ʻo ka ʻāpana N-terminal o ka polypeptide RC-M (e launa pū ana me ka pūnaewele hoʻopaʻa QB ma o ka pilina H o ke koena Tyr ma ka Q helix) e lawe i kahi ʻano kūlohelohe ʻole, a ʻaʻole i ʻimi hou ʻia ka loli ʻano QB (30). ʻO ka mea hōʻoluʻolu, ʻaʻole mākou i ʻike i kēia ʻano deformation o ka polypeptide M i loko o ka RC-LH114-W structure, kahi kokoke like me ka ʻāpana N-terminal o RC-LH116 RC. Pono hoʻi e hoʻomaopopo ʻia ma hope o ka hoʻopau ʻana i ka antenna LH1 i hoʻokumu ʻia i ka detergent, ua hoʻonā ʻia nā apolipoprotein RC i loko o ka PDB, kahi i hoʻopau ai i nā loko quinone kūloko a me nā lipids i loko o ka hakahaka ma waena o ka RC a me ka ʻili o loko o ke apo LH1 a puni (31, 32). Ke hana mau nei ʻo RC no ka mea e mālama ana i nā cofactors āpau, koe wale nō ka QB quinone decomposable, kahi mea paʻa ʻole a nalowale pinepine i ka wā o ke kaʻina hana hoʻomākaukau (33). Eia kekahi, ua ʻike ʻia ʻo ka wehe ʻana o LH1 a me nā lipids cyclic kūlohelohe mai RC hiki ke loaʻa ka hopena i nā hana, e like me ke ola pōkole o ka mokuʻāina P+QB i hoʻokaʻawale ʻia (31, 34, 35). No laila, ke kuhi nei mākou ʻo ke ola ʻana o ke apo LH1 kūloko e hoʻopuni ana i ka RC e mālama paha i ka pūnaewele QB "pani ʻia", no laila e mālama ana i ke kaiapuni kūloko kokoke i ka QB.
ʻOiai ʻo ka apolipoprotein (me ka ʻole o QB quinone) a me ke ʻano piha e hōʻike ana i ʻelua mau kiʻi o ka huli ʻana o ka pūnaewele QB, ma mua o kahi moʻo o nā hanana, aia nā hōʻailona e hiki ke hoʻopaʻa ʻia ka nakinaki e pale i ka hoʻopaʻa hou ʻana e ka hydroquinone E kāohi i ka pale ʻana o ka substrate. ʻOkoʻa paha ka launa pū ʻana o ka quinolol a me ka quinone kokoke i ka pūnaewele QB o ka apolipoprotein, kahi e alakaʻi ai i kona hōʻole ʻia e RC. Ua lōʻihi ka manaʻo ʻia he kuleana ko nā loli conformational i ka nakinaki ʻana a me ka hōʻemi ʻana o nā quinones. Ua hoʻopilikia ʻia ka hiki o nā RC hau e hōʻemi i nā quinones ma hope o ka hoʻololi ʻana i ka pōʻeleʻele (36); Hōʻike ka crystallography X-ray ua hōʻino ʻia kēia pōʻino ma muli o ka hoʻopaʻa ʻia ʻana o nā quinones QB i loko o kahi conformation "distal" ma kahi o 4.5 Å mai ke kūlana proximal hana (26), 37). Manaʻo mākou ʻo kēia conformation nakinaki distal he kiʻi o ke kūlana waena ma waena o ka apolipoprotein a me ke ʻano apo piha, e hahai ana i ka launa mua me ka quinone a me ka wehe ʻana o ka pūnaewele QB.
ʻO ke ʻano II RC i loaʻa i loko o ka PSII complex o kekahi mau bacteria phototrophic a me cyanobacteria, algae a me nā mea kanu he mālama ʻana i ke ʻano a me ka hana (38). ʻO ke kūlike ʻana o ke ʻano i hōʻike ʻia ma ke Kiʻi 6 (D a me E) e hōʻike ana i ka like ma waena o PSII RCs a me kahi QB o ka bacterial RC complex. Ua lōʻihi kēia hoʻohālikelike ʻana i kumu hoʻohālike no ke aʻo ʻana i nā ʻōnaehana pili loa o ka hoʻopaʻa ʻana a me ka hōʻemi ʻana o ka quinone. Ua hōʻike ʻia e nā puke paʻi mua e hele pū ana nā loli conformational me ka hōʻemi ʻana o PSII o nā quinones (39, 40). No laila, i ka noʻonoʻo ʻana i ka mālama ʻana o ka evolutionary o RC, hiki ke pili kēia ʻano hoʻopaʻa i ʻike ʻole ʻia ma mua i ka QB site o PSII RC i nā mea kanu phototrophic oxygenated.
ʻO Rps ΔpufW (ka holoi ʻana o ka pufW me ka lepili ʻole) a me PufW-His (C-terminal 10x His-tagged protein-W i hōʻike ʻia mai ka pufW locus kūlohelohe). ua wehewehe ʻia ʻo palustris CGA009 i kā mākou hana ma mua (16). Ua hoʻihoʻi ʻia kēia mau ʻano a me ka makua wild-type isogenic mai ka pahu hau ma ke kaha ʻana i kahi helu liʻiliʻi o nā cell ma PYE (kēlā me kēia 5 g lita -1) (mālama ʻia i loko o LB ma -80 °C, nona ka 50% (w/v) glycerol) protein, yeast extract a me ka succinate) agar [1.5% (w/v)] pā. Ua hoʻomoʻa ʻia ka pā i ka pō i loko o ka pouli ma ka mahana o ka lumi ma lalo o nā kūlana anaerobic, a laila hoʻomālamalama ʻia me ke kukui keʻokeʻo (~50 μmolm-2 s-1) i hāʻawi ʻia e nā kukui halogen OSRAM 116-W (RS Components, UK) no 3 a 5 mau lā a hiki i ka wā i ʻike ʻia ai kahi kolone hoʻokahi. Ua hoʻohana ʻia kahi kolone hoʻokahi e hoʻokomo i 10 ml o ka M22+ medium (41) i hoʻohui ʻia me 0.1% (w/v) casamino acids (ma hope aku i kapa ʻia ʻo M22). Ua ulu ʻia ka moʻomeheu ma lalo o nā kūlana oxygen haʻahaʻa i ka pōʻeleʻele ma 34°C me ka lulu ʻana ma 180 rpm no 48 mau hola, a laila ua hoʻokomo ʻia he 70 ml o ka moʻomeheu ma lalo o nā kūlana like no 24 mau hola. Hoʻohana ʻia kahi moʻomeheu semi-aerobic me ka nui o 1 ml e hoʻokomo i 30 ml o ka M22 medium i loko o kahi ʻōmole aniani aniani wili-luna 30 ml a hoʻomālamalama ʻia me ka hoʻoulu ʻana (~50μmolm-2 s-1) no 48 mau hola e ka ikaika magnetic sterile Stirring rod. A laila ua hoʻokomo ʻia he 30 ml o ka moʻomeheu me kahi 1 lita o ka moʻomeheu ma lalo o nā kūlana like, a laila ua hoʻohana ʻia e hoʻokomo i kahi 9 lita o ka moʻomeheu i hoʻomālamalama ʻia ma ~200 μmolm-2 s-1 no 72 mau hola. Ua hōʻiliʻili ʻia nā pūnaewele ma o ka centrifugation ma 7132 RCF no 30 mau minuke, hoʻoulu hou ʻia i loko o ~10 ml o 20 mM tris-HCl (pH 8.0), a mālama ʻia ma -20°C a hiki i ka wā e pono ai.
Ma hope o ka hoʻoheheʻe ʻana, e hoʻohui i kekahi mau kristal o deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) a me ʻelua mau papa Roche holoenzyme protease inhibitor (Merck, UK) i nā cell i hoʻoulu hou ʻia. I loko o kahi cell kaomi Farani 20,000 psi (Aminco, USA), ua hoʻopilikia ʻia nā cell i 8 a 12 mau manawa. Ma hope o ka wehe ʻana i nā cell i haki ʻole ʻia a me nā ʻōpala hiki ʻole ke hoʻoheheʻe ʻia ma ka centrifugation ma 18,500 RCF no 15 mau minuke ma 4°C, ua hoʻoheheʻe ʻia ka membrane mai ka lysate pigmented ma ka centrifugation ma 113,000 RCF no 2 mau hola ma 43,000°C. E hoʻolei i ka ʻāpana hiki ke hoʻoheheʻe ʻia a hoʻoulu hou i ka membrane kala i loko o 100 a 200 ml o 20 mM tris-HCl (pH 8.0) a homogenize a hiki i ka wā ʻaʻohe aggregates ʻike ʻia. Ua hoʻomoʻa ʻia ka membrane i hoʻokuʻu ʻia i loko o 20 mM tris-HCl (pH 8.0) (Anatrace, USA) nona ka 2% (w/v) β-DDM no 1 hola i ka pouli ma 4°C me ka hoʻowali mālie. A laila centrifuge ma 70°C e hoʻoheheʻe i 150,000 RCF ma 4°C no 1 hola e wehe i nā mea insolubles i koe.
Ua hoʻopili ʻia ka membrane hoʻoheheʻe mai ke ʻano ΔpufW i kahi kolamu hoʻololi ion DEAE Sepharose 50 ml me ʻekolu mau leo ​​kolamu (CV) o ka buffer hoʻopaʻa [20 mM tris-HCl (pH 8.0) nona ka 0.03% (w / v) β-DDM]. E holoi i ke kolamu me ʻelua mau buffer hoʻopaʻa CV, a laila holoi i ke kolamu me ʻelua mau buffer hoʻopaʻa e loaʻa ana he 50 mM NaCl. Ua hoʻoheheʻe ʻia ka complex RC-LH116 me kahi gradient linear o 150 a 300 mM NaCl (i loko o ka buffer hoʻopaʻa) ma 1.75 CV, a ua hoʻoheheʻe ʻia ke koena o ka complex hoʻopaʻa me kahi buffer hoʻopaʻa e loaʻa ana he 300 mM NaCl ma 0.5 CV. E hōʻiliʻili i ka spectrum absorption ma waena o 250 a me 1000 nm, e mālama i ka ʻāpana me ka lakio absorbance (A880/A280) ʻoi aku ma mua o 1 ma 880 a 280 nm, e hoʻoheheʻe iā ia i ʻelua manawa i loko o ka buffer binding, a hoʻohana hou i ke kaʻina hana like ma ke kolamu DEAE Ma ka hoʻomaʻemaʻe. E hoʻoheheʻe i nā ʻāpana me nā lakio A880/A280 ʻoi aku ma mua o 1.7 a me nā lakio A880/A805 ʻoi aku ma mua o 3.0, e hana i ke kolu o ka puni o ka hoʻololi ion, a mālama i nā ʻāpana me nā lakio A880/A280 ʻoi aku ma mua o 2.2 a me nā lakio A880/A805 ʻoi aku ma mua o 5.0. Ua hoʻopili ʻia ka hui i hoʻomaʻemaʻe hapa ʻia i ~2 ml i loko o kahi kānana centrifugal Amicon 100,000 molecular weight cut-off (MWCO) (Merck, UK), a ua hoʻouka ʻia ma luna o kahi kolamu exclusion Superdex 200 16/600 size (GE Healthcare, US) nona ka buffer 200 mM NaCl, a laila eluted i loko o ka buffer like ma 1.5 CV. E hōʻiliʻili i nā spectra absorption o ka size exclusion fraction, a hoʻopili i nā spectra absorption me nā lakio A880/A280 ma luna o 2.4 a me nā lakio A880/A805 ma luna o 5.8 a 100 A880, a hoʻohana koke iā lākou no ka hoʻomākaukau ʻana a i ʻole ka mālama ʻana i ka grid cryo-TEM E mālama ma -80°C a hiki i ka wā e pono ai.
Ua hoʻopili ʻia ka membrane solubilizing mai ka PufW-His strain i kahi kolamu 20 ml HisPrep FF Ni-NTA Sepharose (20 mM tris-HCl (pH 8.0) e loaʻa ana he 200 mM NaCl a me 0.03% (w/w)) i loko o ka buffer IMAC (GE Healthcare). v) β-DDM]. Ua holoi ʻia ke kolamu me ʻelima CV o ka buffer IMAC, a laila me ʻelima CV o ka buffer IMAC e loaʻa ana he 10 mM histidine. Ua hoʻokaʻawale ʻia ka hui kumu mai ke kolamu me ʻelima mau buffer IMAC e loaʻa ana he 100 mM histidine. Hoʻopili ʻia ka hapa e loaʻa ana ka hui RC-LH114-W i ~10 ml i loko o kahi pahu i hoʻoulu ʻia me kahi kānana Amicon 100,000 MWCO (Merck, UK), hoʻokahe ʻia he 20 mau manawa me ka buffer nakinaki, a laila hoʻohui ʻia i 25 ml I loko o ke kolamu DEAE Sepharose, ʻehā mau CV i hoʻopaʻa ʻia i ka buffer i hoʻohana mua ʻia. E holoi i ke kolamu me ʻehā mau mea hoʻopaʻa CV, a laila elute i ka complex ma nā CV ʻewalu ma kahi gradient linear o 0 a 100 mM NaCl (i loko o ka buffer binding), a me nā CV ʻehā i koe me 100 mM binding buffer. ʻO nā complexes koena i eluted ma ka sodium chloride i hui pū ʻia me ka ratio A880/A280 ʻoi aku ka kiʻekiʻe ma mua o 2.4 a me ka ratio A880/A805 ʻoi aku ka kiʻekiʻe ma mua o 4.6 mau ʻāpana i hoʻopaʻa ʻia i ~2 ml i loko o kahi kānana centrifugal Amicon 100,000 MWCO, a hoʻopiha ʻia me 1.5 CV IMAC ma mua. Ua hoʻohālikelike ʻo Buffer i ka Superdex 200 16/600 size exclusion column, a laila eluted i loko o ka buffer like ma luna o 1.5 CV. E hōʻiliʻili i nā spectra absorption o nā ʻāpana hoʻokaʻawale-nui a hoʻohuihui i nā spectra absorption me nā lakio A880/A280 ma luna o 2.1 a me nā lakio A880/A805 ma luna o 4.6 a 100 A880, kahi i hoʻohana koke ʻia no ka hoʻomākaukau ʻana i ka grid TEM hau a mālama ʻia paha ma -80°C a hiki i ka wā e pono ai.
Ua hoʻohana ʻia kahi pahu hau hoʻoluʻu Leica EM GP e hoʻomākaukau i nā grids TEM haʻahaʻa haʻahaʻa. Ua hoʻoheheʻe ʻia ka paʻakikī i loko o ka buffer IMAC i A880 o 50, a laila ua hoʻouka ʻia he 5μl ma luna o kahi ʻupena keleawe QUANTIFOIL 1.2/1.3 i uhi ʻia i ke kalapona (Agar Scientific, UK). E hoʻomoʻa i ka grid ma 20°C a me 60% ka haʻahaʻa pili no 30 s, a laila e holoi maloʻo no 3 s, a laila e kinai iā ia i loko o ka ethane wai ma -176°C.
Ua hoʻopaʻa ʻia ka ʻikepili o ka paʻakikī RC-LH114-W ma ka eBIC (Electronic Bioimaging Center) (British Diamond Light Source) me kahi microscope Titan Krios, kahi e hana ana ma kahi volta wikiwiki o 300kV, me ka hoʻonui inoa o 130,000 × a me ka ikehu o- E koho i kahi hakahaka o 20 eV. Ua hoʻohana ʻia kahi Gatan 968 GIF Quantum me ka mea ʻike piko K2 e hoʻopaʻa i nā kiʻi ma ke ʻano helu e hōʻiliʻili i ka ʻikepili. ʻO ka nui o ka pixel i hoʻoponopono ʻia he 1.048Å, a ʻo ka helu mahele he 3.83 e-Å-2s-1. Ua hōʻiliʻili i ke kiʻiʻoniʻoni i loko o 11 kekona a ua māhele ʻia i 40 mau ʻāpana. E hoʻohana i ka ʻāpana i uhi ʻia i ke kalapona e hoʻohuli hou i ka microscope, a laila e hōʻiliʻili i ʻekolu mau kiʻiʻoniʻoni no kēlā me kēia lua. Ma ka huina, ua hōʻiliʻili ʻia he 3130 mau kiʻiʻoniʻoni, me nā waiwai defocus ma waena o -1 a me -3μm.
Ua hōʻiliʻili ʻia ka ʻikepili no ka paʻakikī RC-LH116 me ka hoʻohana ʻana i ka microscope like ma ka Asterbury Biostructure Laboratory (University of Leeds, UK). Ua hōʻiliʻili ʻia ka ʻikepili ma ke ʻano helu me ka hoʻonui ʻana o 130 k, a ua hoʻoponopono ʻia ka nui o ka pixel i 1.065 Å me ka nui o 4.6 e-Å-2s-1. Ua hoʻopaʻa ʻia ke kiʻiʻoniʻoni i loko o 12 kekona a ua māhele ʻia i 48 mau ʻāpana. Ma ka huina, ua hōʻiliʻili ʻia he 3359 mau kiʻiʻoniʻoni, me nā waiwai defocus ma waena o -1 a me -3μm.
Hana ʻia nā hana ʻikepili āpau ma ka pipeline Relion 3.0 (42). E hoʻohana iā Motioncorr 2 (43) e hoʻoponopono i ka neʻe ʻana o ke kukuna ma o ke kaupaona ʻana i ka lāʻau, a laila e hoʻohana iā CTFFIND 4.1 (44) e hoʻoholo i ka palena CTF (hana hoʻoili ʻokoʻa). Hōʻike ʻia nā kiʻi photomicrograph maʻamau ma hope o kēia mau pae hana mua ma ke Kiʻi 2. S16. Hoʻokumu ʻia ka template koho aunoa ma ke koho lima ʻana ma kahi o 250 mau pikela o 1000 mau ʻāpana i loko o kahi kiʻi 250-pikela a ʻaʻohe hoʻokaʻawale ʻelua-dimensional (2D), no laila e hōʻole ana i kēlā mau hoʻokaʻawale e kūpono i ka haumia o ka laʻana a i ʻole ʻaʻohe ʻano ʻike. A laila, ua hana ʻia ke koho aunoa ma nā microphotograph āpau, a ʻo ka RC-LH114-W he 849,359 mau ʻāpana, a ʻo ka paʻakikī RC-LH116 he 476,547 mau ʻāpana. Ua hala nā ʻāpana āpau i koho ʻia i ʻelua mau puni o ka hoʻokaʻawale ʻana o 2D ʻaʻole kuhikuhi, a ma hope o kēlā me kēia holo, ua hōʻole ʻia nā ʻāpana e kū ana i ka ʻāpana kalapona, ka haumia o ka laʻana, ʻaʻohe hiʻohiʻona maopopo a i ʻole nā ​​​​ʻāpana e pili pū ana, e hopena ana i 772,033 (90.9%) a me 359,678 (75.5%) ) Hoʻohana ʻia nā ʻāpana no ka hoʻokaʻawale ʻana o RC-LH114-W a me RC-LH116. Ua hana ʻia ke kumu hoʻohālike kuhikuhi 3D mua me ka hoʻohana ʻana i ke ʻano hoʻohaʻahaʻa gradient stochastic. Ma ka hoʻohana ʻana i ke kumu hoʻohālike mua ma ke ʻano he kuhikuhi, ua hoʻokaʻawale ʻia nā ʻāpana i koho ʻia i ʻehā mau māhele ma 3D. Ma ka hoʻohana ʻana i ke kumu hoʻohālike ma kēia māhele ma ke ʻano he kuhikuhi, e hana i ka hoʻomaʻemaʻe 3D ma nā ʻāpana ma ka māhele nui loa, a laila e hoʻohana i ka kānana haʻahaʻa haʻahaʻa 15Å mua e uhi i ka ʻāpana solvent, e hoʻohui i 6 mau pikela o nā ʻaoʻao palupalu, a ma hope o ke kaʻina hana ʻana i nā pikela e hoʻoponopono i ka hana hoʻoili Modulation Gatan K2 peak o ka mea ʻike luna. No ka ʻikepili RC-LH114-W, ua hoʻololi ʻia kēia kumu hoʻohālike mua ma ka wehe ʻana i ka nui ikaika ma nā kihi o ka pale maka (i hoʻokaʻawale ʻia mai ka nui paʻakikī koʻikoʻi ma UCSF Chimera). ʻO nā kumu hoʻohālike hopena (ʻo nā hoʻonā o RC-LH114-W a me RC-LH116 he 3.91 a me 4.16 Å, kēlā me kēia) ua hoʻohana ʻia ma ke ʻano he kuhikuhi no ka lua o ka puni o ka hoʻokaʻawale ʻana 3D. Hoʻohui ʻia nā ʻāpana i hoʻohana ʻia i loko o ka papa 3D mua a ʻaʻohe pilina ikaika me ka kaiāulu. Hoʻopili a nele paha i nā hiʻohiʻona kūkulu maopopo. Ma hope o ka lua o ka puni o ka hoʻokaʻawale ʻana 3D, ua koho ʻia ka māhele me ka hoʻonā kiʻekiʻe loa [No RC-LH114-W, hoʻokahi māhele he 377,703 mau ʻāpana (44.5%), no RC-LH116, aia ʻelua mau māhele, he 260,752 mau ʻāpana (54.7%), kahi like lākou ke hoʻonohonoho ʻia ma hope o ka hoʻohuli mua me kahi ʻokoʻa liʻiliʻi]. Hoʻopuka hou ʻia nā ʻāpana i koho ʻia i loko o kahi pahu 400-pixel a hoʻomaʻemaʻe ʻia e ka hoʻomaʻemaʻe 3D. Hoʻokumu ʻia ka pale maka solvent me ka hoʻohana ʻana i ka kānana haʻahaʻa mua 15Å, ka hoʻonui ʻana o ka palapala ʻāina 3 pixel a me ka pale maka palupalu 3 pixel. Ma ka hoʻohana ʻana i ka hoʻomaʻemaʻe ʻana o ka CTF per-particle, ka hoʻoponopono ʻana i ka neʻe ʻana o kēlā me kēia ʻāpana a me ka lua o ka pōʻai o ka hoʻomaʻemaʻe ʻana o ka CTF per-particle, ka hoʻomaʻemaʻe ʻana o 3D, ka pale maka solvent a me ka hana hope e hana ʻia ma hope o kēlā me kēia ʻanuʻu e hoʻomaʻemaʻe hou aku i ke ʻano hopena. Ma ka hoʻohana ʻana i ka waiwai ʻoki FSC (Fourier Shell Correlation Coefficient) o 0.143, ʻo nā hoʻonā o nā hiʻohiʻona hope loa o RC-LH114-W a me RC-LH116 he 2.65 a me 2.80Å, kēlā me kēia. Hōʻike ʻia ka piʻo FSC o ke kumu hoʻohālike hope loa ma ke Kiʻi 2. S17.
Hoʻoiho ʻia nā moʻo protein āpau mai 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). Ua hoʻohana ʻia ʻo SWISS-MODEL (45) e kūkulu i kahi kumu homology o RC, nona nā moʻo protein o RC-L, RC-M a me RC-H a me ke ʻano kristal o Rba. Ua hoʻohana ʻia ʻo sphaeroides RC ma ke ʻano he template (PDB ID: 5LSE) (46). E hoʻohana i ka mea hana "fit map" ma UCSF Chimera e hoʻokomo i ke kumu hoʻohālike i hana ʻia i ka palapala ʻāina (47), hoʻomaikaʻi i ke ʻano o ka protein, a me ka cofactor [4×BChl a (inoa koena waihona puke monomer = BCL), 2×BPh a (BPH), hoʻokahi a ʻelua paha ʻano UQ10 (U10), hoʻokahi hao non-heme (Fe) a me hoʻokahi 3,4-dihydrohexacarbonylcholine (QAK)] e hoʻohana iā Coot (48) e hoʻohui. ʻOiai ʻaʻole loaʻa ʻo QAK i loko o ka waihona puke monomer, ua hoʻohālikelike ʻia me ka hoʻohana ʻana i ka mea hana eLBOW ma PHENIX (49).
ʻO ka mea aʻe, ua kūkulu ʻia ka subunit LH1. I ka wā mua, ua hoʻohana ʻia ka mea hana kūkulu ʻakomi ma PHENIX (49) e kūkulu ʻakomi i kahi ʻāpana o ka moʻo LH1 me ka hoʻohana ʻana i ka palapala ʻāina a me nā moʻo protein LH1-α a me LH1-β ma ke ʻano he mea hoʻokomo. E koho i ka subunit LH1 piha loa, e unuhi iā ia a hoʻouka iā ia i loko o Coot, e hoʻohui lima i ka moʻo i nalowale i loko, a hoʻomaʻemaʻe lima i ka ʻōnaehana holoʻokoʻa ma mua o ka hoʻohui ʻana i ʻelua BCls a (BCL) a me kahi spirilloxanthin (CRT) [e like me nā Rps pili ʻO ka nui o ka paʻakikī LH1 a me ka ʻike carotenoid i ʻike ʻia. ʻAno (17)]. E kope i ka subunit LH1 piha, a e hoʻohana i ka UCSF Chimera "Docking Map Tool" e hoʻopaʻa i ka ʻāpana non-model e pili ana o ka nui o LH1, a laila hoʻomaʻemaʻe iā ia ma Coot; e hana hou i ke kaʻina hana a hiki i ka hoʻohālike ʻia ʻana o nā subunit LH1 āpau. No ka hoʻonohonoho ʻana o RC-LH114-W, ma ka unuhi ʻana i ka nui i hoʻokaʻawale ʻole ʻia ma Coot, ua hoʻokaʻawale ʻia ka protein mai nā ʻāpana protein ʻole i koe ma ka palapala ʻāina USCF Chimera a hoʻohana ʻia ka mea hana Autobuild e hoʻokumu i ke kumu hoʻohālike mua, a me nā subunits i koe (protein-W) Modeling. Ma PHENIX (49). E hoʻohui i kekahi mau kaʻina i nalowale i ke kumu hoʻohālike hopena ma Coot (48), a laila e hoʻomaʻemaʻe lima i ka subunit holoʻokoʻa. ʻO ke koena o ka nui i hoʻokaʻawale ʻole ʻia e kūpono i ka hui pū ʻana o nā lipids (PDB monomer library ID o CDL = CDL, POPC = 6PL a me POPG = PGT), β-DDM detergent (LMT) a me nā molekala UQ10 (U10). E hoʻohana i ka PHENIX optimization (49) a me ka manual optimization ma Coot (48) e hoʻomaikaʻi i ke kumu hoʻohālike mua piha a hiki i ka hiki ʻole ke hoʻomaikaʻi hou ʻia nā helu kumu hoʻohālike a me ke ʻano ʻike o ke kūpono. ʻO ka hope loa, e hoʻohana iā LocScale (50) e hoʻopololei i ka palapala ʻāina kūloko, a laila e hana i kekahi mau pōʻaiapuni ʻē aʻe o ke kumu hoʻohālike i ka nui i hoʻokaʻawale ʻole ʻia a me ka manual optimization aunoa a me ka manual.
Ua hōʻike ʻia nā peptides, cofactors a me nā lipids a me nā quinones ʻē aʻe i hoʻopaʻa ʻia i loko o ko lākou mau densities ma nā Kiʻi 1 a me 2. S18 a i S23. Ua hōʻike ʻia ka ʻike helu o ke kumu hoʻohālike hope loa ma ka Papa S1.
Inā ʻaʻole i kuhikuhi ʻia, ua hōʻiliʻili ʻia nā spectra absorption UV/Vis/NIR ma kahi spectrophotometer Cary60 (Agilent, USA) ma nā manawa 1 nm mai 250 nm a 1000 nm a me kahi manawa hoʻohui o 0.1s.
E hoʻoheheʻe i ka hāpana i loko o kahi cuvette quartz me ke ala 2 mm i A880 o 1, a hōʻiliʻili i ka spectrum absorption ma waena o 400 a me 1000 nm. Ua hōʻiliʻili ʻia nā spectra dichroic circular ma kahi spectropolarimeter Jasco 810 (Jasco, Iapana) ma nā manawa 1 nm ma waena o 400 nm a me 950 nm ma ka helu scan o 20 nm min-1.
Hoʻoholo ʻia ke koina hoʻopau molar ma ka hoʻoheheʻe ʻana i ka paʻakikī kumu i kahi A880 ma kahi o 50. E hoʻoheheʻe i ka nui o 10μl i loko o ka buffer nakinaki 990μl a i ʻole methanol, a hōʻiliʻili koke i ka spectrum absorption e hōʻemi i ka hoʻohaʻahaʻa ʻana o BChl. Ua helu ʻia ka nui o BChl o kēlā me kēia laʻana methanol e ka coefficient extinction ma 771 nm o 54.8 mM-1 cm-1, a ua hoʻoholo ʻia ke koina extinction (51). E puʻunaue i ka nui o BChl i ana ʻia e 32 (RC-LH114-W) a i ʻole 36 (RC-LH116) e hoʻoholo i ka nui o ka paʻakikī kumu, a laila hoʻohana ʻia e hoʻoholo i ka spectrum absorption o ka laʻana like i hōʻiliʻili ʻia ma ka buffer Extinction coefficient. ʻEkolu mau ana i hana hou ʻia no kēlā me kēia laʻana, a ua hoʻohana ʻia ka awelika absorbance o ka BChl Qy maximum no ka helu ʻana. ʻO ke koina hoʻopau ʻana o RC-LH114-W i ana ʻia ma 878 nm he 3280±140 mM-1 cm-1, ʻoiai ʻo ke koina hoʻopau ʻana o RC-LH116 i ana ʻia ma 880 nm he 3800±30 mM-1 cm-1.
Ua helu ʻia ʻo UQ10 e like me ke ʻano ma (52). I ka pōkole, ua hana ʻia ka HPLC pae hoʻohuli (RP-HPLC) me ka hoʻohana ʻana i ka ʻōnaehana Agilent 1200 HPLC. E hoʻoheheʻe ma kahi o 0.02 nmol o RC-LH116 a i ʻole RC-LH114-W i loko o 50μl o 50:50 methanol:chloroform e loaʻa ana he 0.02% (w/v) ferric chloride, a e hoʻokomo i ka Beckman Coulter Ultrasphere ODS 4.6 mm i hoʻohālikelike mua ʻia. Hoʻoheheʻe ʻia i loko o 1 ml-1 min-1 ma 40°C i loko o ka mea hoʻoheheʻe HPLC (80:20 methanol:2-propanol) ma kahi kolamu ×25 cm. E hana i ka elution isocratic i loko o kahi mea hoʻoheheʻe HPLC e nānā i ka absorbance ma 275 nm (UQ10), 450 nm (carotenoids) a me 780 nm (BChl) no 1 hola. Ua hoʻohui ʻia ka piko i loko o ka chromatogram 275 nm ma 25.5 mau minuke, ʻaʻohe ona hui ʻike ʻē aʻe. Hoʻohana ʻia ka ʻāpana i hoʻohui ʻia e helu i ka nui molar o UQ10 i unuhi ʻia me ka ʻōlelo ʻana i ke kaʻina hoʻoponopono i helu ʻia mai ka hoʻokomo ʻana o nā kūlana maʻemaʻe mai 0 a 5.8 nmol (Kiʻi S14). Ua kālailai ʻia kēlā me kēia laʻana i ʻekolu mau replicates, a ʻo ka hewa i hōʻike ʻia e pili ana i ka SD o ka awelika.
Ua hoʻomākaukau ʻia kahi hopena e loaʻa ana ka hui RC-LH1 me ka absorption Qy kiʻekiʻe loa o 0.1 me 30 μM cytochrome puʻuwai lio i hoʻemi ʻia c2 (Merck, UK) a me 0 a 50 μMUQ2 (Merck, UK). Ua hoʻomākaukau ʻia ʻekolu mau laʻana 1-ml ma kēlā me kēia ʻano UQ2 a hoʻomoʻa ʻia i ka pō i loko o ka pouli ma 4°C e hōʻoia i ka hoʻololi piha ʻana i ka pouli ma mua o ke ana ʻana. Ua hoʻouka ʻia ka hopena i loko o kahi spectrophotometer modular OLIS RSM1000 i lako me kahi grating laina lapalapa 300 nm/500, kahi komo 1.24 mm, kahi waena 0.12 mm a me nā ʻāpana puka 0.6 mm. Ua kau ʻia kahi kānana pass lōʻihi 600 nm ma ke komo ʻana o ka phototube laʻana a me ka paipu photomultiplier kuhikuhi e kāpae i ka mālamalama hoʻonāukiuki. Ua nānā ʻia ka absorbance ma 550 nm me ka manawa hoʻohui o 0.15 s. Hoʻopuka ʻia ke kukui hoʻonāukiuki mai ka 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) ma o ke kaula fiber optic ma ka ikaika 90% ma o kahi mea hoʻokele DC2200 (Thorlabs Ltd., UK) a hoʻopuka ʻia i ke kumu kukui ma kahi kihi o 90° o. Kūʻē ke kukuna ana i ke aniani e hoʻihoʻi i kekahi kukui i komo ʻole ʻia e ka laʻana. E nānā i ka absorbance 10 s ma mua o ka hoʻomālamalama ʻana o 50 s. A laila ua nānā hou ʻia ka absorbance no 60 s i ka pōʻeleʻele e loiloi i ka nui o ka quinolol e hōʻemi koke i ka cytochrome c23 + (e nānā i ke Kiʻi S8 no ka ʻikepili maka).
Ua hana ʻia ka ʻikepili ma ke kau ʻana i kahi helu hoʻomaka linear i loko o 0.5 a 10 s (ma muli o ka nui o ka UQ2) a me ka awelika ʻana i nā helu o nā laʻana ʻekolu ma kēlā me kēia nui o ka UQ2. Ua hoʻohana ʻia ka nui o ka RC-LH1 i helu ʻia e ka coefficient extinction e hoʻololi i ka helu i ka pono catalytic, i hoʻolālā ʻia ma Origin Pro 2019 (OriginLab, USA), a ua hoʻopili ʻia i ke kumu hoʻohālike Michaelis-Menten e hoʻoholo i nā waiwai Km a me Kcat i ʻike ʻia.
No nā ana omo transient, ua hoʻoheheʻe ʻia ka hāpana RC-LH1 i ~ 2μM i loko o ka buffer IMAC e loaʻa ana he 50 mM sodium ascorbate (Merck, USA) a me 0.4 mM Terbutin (Merck, USA). Hoʻohana ʻia ka waikawa Ascorbic ma ke ʻano he mea hāʻawi electron mōhai, a hoʻohana ʻia ka tert-butaclofen ma ke ʻano he mea hoʻopaʻa QB e hōʻoia i ka emi ʻana o ka mea hāʻawi RC nui (ʻo ia hoʻi, ʻaʻole i photooxidized) ma ke kaʻina hana ana. Ma kahi o 3 ml o ka hāpana i hoʻohui ʻia i kahi cell rotating maʻamau (ma kahi o 0.1 m ke anawaena, 350 RPM) me ka lōʻihi o ke ala optical 2 mm e hōʻoia i ka lawa ʻana o ka manawa o ka hāpana ma ke ala laser no ka hoʻololi ʻana i ka pōʻeleʻele ma waena o nā pulse excitation. E hoʻohana i nā pulse laser ~ 100-fs e hoʻonui i ka ʻōnaehana laser Ti: Sapphire (Spectra Physics, USA) e hoʻonāukiuki i ka hāpana ma 880 nm ma ka helu hana hou o 1 kHz (20 nJ no NIR a i ʻole 100 nJ no Vis). Ma mua o ka hōʻiliʻili ʻana i ka ʻikepili, e hōʻike i ka hāpana i ka mālamalama hoʻonāukiuki no kahi o 30 mau minuke. ʻO ka hōʻike ʻana e hoʻopau ai i ka QA (hiki ke hōʻemi i ka QA i hoʻokahi a ʻelua paha). Akā e ʻoluʻolu e hoʻomaopopo he hiki ke hoʻohuli ʻia kēia kaʻina hana no ka mea ma hope o kahi manawa lōʻihi o ka hoʻololi ʻana i ka pouli, e hoʻi mālie ʻo RC i ka hana QA. Ua hoʻohana ʻia kahi spectrometer Helios (Ultrafast Systems, USA) e ana i nā spectra transient me ka manawa lohi o -10 a 7000 ps. E hoʻohana i ka polokalamu Surface Xplorer (Ultrafast Systems, USA) e hoʻokaʻawale i nā ʻikepili, a laila hoʻohui a hoʻonohonoho. E hoʻohana i ka pūʻolo polokalamu CarpetView (Light Conversion Ltd., Lithuania) e hoʻohana i ka ʻikepili i hui pū ʻia e loaʻa nā spectra ʻokoʻa e pili ana i ka palaho, a i ʻole e hoʻohana i kahi hana e hoʻohui i nā exponents he nui me ka pane o ka mea hana e kūpono i ka evolution spectral single-wavelength ma Origin (OriginLab, USA).
E like me ka mea i ʻōlelo ʻia ma luna (53), ua hoʻomākaukau ʻia kahi kiʻiʻoniʻoni photosynthetic e loaʻa ana ka paʻakikī LH1 nele i ka RC a me ka peripheral LH2 antenna. Ua hoʻoheheʻe ʻia ka membrane i loko o 20 mM tris (pH 8.0) a laila ua hoʻouka ʻia i loko o kahi cuvette quartz me kahi ala optical 2 mm. Ua hoʻohana ʻia kahi pulse laser 30nJ e hoʻonāukiuki i ka laʻana ma 540 nm me ka manawa lohi o -10 a 7000 ps. E hana i ka ʻikepili i hoʻonohonoho ʻia e like me ka mea i wehewehe ʻia no Rps. pal sample.
Ua pellet ʻia ka membrane ma o ka centrifugation ma 150,000 RCF no 2 mau hola ma 4°C, a laila ua hoʻoulu hou ʻia kona absorbance ma 880 nm i loko o 20 mM tris-HCl (pH 8.0) a me 200 mM NaCl. E hoʻoheheʻe i ka membrane ma ka hoʻoulu mālie ʻana i ka 2% (w/v) β-DDM no 1 hola i ka pōʻeleʻele ma 4°C. Ua hoʻoheheʻe ʻia ka laʻana i loko o 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) i kahi ʻano protein o 2.5 mg ml-1 (Bio-Rad analysis). Ua hana ʻia ka hana hou ʻana mai ke ʻano i paʻi mua ʻia (54), e hoʻomaka ana me ka dilution o 50 μg protein i loko o ka huina o 50 μl TEAB e loaʻa ana he 1% (w/v) sodium laurate (Merck, UK). Ma hope o ke sonication no 60 s, ua hoʻemi ʻia me 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) ma 37°C no 30 mau minuke. No ka S-alkylation, e hoʻomoʻa i ka hāpana me 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) a hoʻohui iā ia mai kahi hopena waihona isopropanol 200 mM no 10 mau minuke ma ka mahana o ka lumi. Ua hoʻokō ʻia ka ʻeli ʻana o ka proteolytic ma ka hoʻohui ʻana i ka hui ʻana o 2 μg trypsin/endoproteinase Lys-C (Promega UK) a hoʻomoʻa ʻia ma 37°C no 3 mau hola. Ua unuhi ʻia ka laurate surfactant ma ka hoʻohui ʻana i 50 μl ethyl acetate a me 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) a me ka vortexing no 60 s. Ua hoʻolaha ʻia ka hoʻokaʻawale ʻana o ka pae ma o ka centrifugation ma 15,700 RCF no 5 mau minuke. Wahi a ke kaʻina hana a ka mea hana, ua hoʻohana ʻia kahi kolamu milo C18 (Thermo Fisher Scientific, UK) e omo pono a wehe i ka paʻakai o ka pae haʻahaʻa e loaʻa ana ka peptide. Ma hope o ka maloʻo ʻana ma o ka centrifugation vacuum, ua hoʻoheheʻe ʻia ka hāpana i loko o 0.5% TFA a me 3% acetonitrile, a ua kālailai ʻia ʻo 500 ng e ka nanoflow RP chromatography i hui pū ʻia me ka mass spectrometry me ka hoʻohana ʻana i nā palena ʻōnaehana i hōʻike mua ʻia.
E hoʻohana iā MaxQuant v.1.5.3.30 (56) no ka ʻike ʻana i ka protein a me ka helu ʻana e ʻimi ai i ka waihona ʻikepili proteome Rps. palustris (www.uniprot.org/proteomes/UP000001426). Ua waiho ʻia ka ʻikepili proteomics mass spectrometry i loko o ka ProteomeXchange Alliance ma o ka waihona hoa PRIDE (http://proteomecentral.proteomexchange.org) ma lalo o ka mea hōʻike dataset PXD020402.
No ka nānā ʻana e RPLC i hui pū ʻia me ka electrospray ionization mass spectrometry, ua hoʻomākaukau ʻia ka RC-LH1 complex mai nā Rps wild-type. Ma ka hoʻohana ʻana i ke ʻano i paʻi mua ʻia (16), ʻo ka nui o ka protein i hana ʻia i loko o nā cell palustris he 2 mg ml-1 i loko o 20 mM Hepes (pH 7.8), 100 mM NaCl a me 0.03% (w/v) β- (Bio-Rad analysis)) DDM. Wahi a ke kaʻina hana a ka mea hana, e hoʻohana i ka pahu hoʻomaʻemaʻe 2D (GE Healthcare, USA) e unuhi i ka 10 μg protein ma ke ʻano precipitation, a hoʻoheheʻe i ka precipitate i loko o 20 μl 60% (v/v) formic acid (FA), 20% (v/v) Acetonitrile a me 20% (v/v) wai. ʻElima microliters i kālailai ʻia e RPLC (Dionex RSLC) i hui pū ʻia me ka mass spectrometry (Maxis UHR-TOF, Bruker). E hoʻohana i ke kolamu MabPac 1.2 × 100 mm (Thermo Fisher Scientific, UK) no ka hoʻokaʻawale ʻana ma 60 °C a me 100 μlmin -1, me kahi gradient o 85% (v / v) solvent A [0.1% (v / v) FA a me 0.02% (V/v) TFA aqueous solution] i 85% (v/v) solvent B [0.1% (v/v) FA a me 0.02% (v/v) ma 90% (v/v) acetonitrile TFA] Ma ka hoʻohana ʻana i ke kumu ionization electrospray maʻamau a me nā palena paʻamau no nā minuke he 60 a ʻoi, loaʻa i ka spectrometer mass 100 a 2750 m/z (mass-to-charge ratio). Me ke kōkua o ka puka kumuwaiwai bioinformatics ExPASy FindPept tool (https://web.expasy.org/findpept/), e palapala i ka spectrum mass i nā subunits o ka complex.
Ua ulu ʻia nā ʻāpana no 72 mau hola ma lalo o 100 ml NF-haʻahaʻa (10μMm-2 s-1), waena (30μMm-2 s-1) a i ʻole kiʻekiʻe (300μMm-2 s-1) mālamalama. M22 waena (M22 waena kahi i kāpae ʻia ai ka ammonium sulfate a ua pani ʻia ka sodium succinate e ka sodium acetate) i loko o kahi ʻōmole wili-luna 100 ml (23). I loko o ʻelima mau pōʻaiapuni 30-s, ua hoʻopili ʻia nā momi aniani 0.1 micron ma ka lakio nui o 1:1 e lyse i nā ʻāpana a hoʻomaʻalili ʻia ma luna o ka hau no 5 mau minuke. Ua wehe ʻia ka mea hiki ʻole ke hoʻoheheʻe ʻia, nā ʻāpana i haki ʻole a me nā momi aniani ma o ka centrifugation ma 16,000 RCF no 10 mau minuke i loko o kahi microcentrifuge benchtop. Ua hoʻokaʻawale ʻia ka membrane i loko o kahi rotor Ti 70.1 me 100,000 RCF i loko o 20 mM tris-HCl (pH 8.0) me kahi gradient sucrose 40/15% (w/w) no 10 mau hola.
E like me ka mea i wehewehe ʻia ma kā mākou hana ma mua, ʻo ka immunodetection o ka tag His ma PufW (16). I ka pōkole, ʻo ka hui kumu i hoʻomaʻemaʻe ʻia (11.8 nM) a i ʻole ka membrane e loaʻa ana ka nui like o RC (i hoʻoholo ʻia e ka oxidation e unuhi ana i ka spectrum ʻokoʻa i hoʻemi ʻia a me ke kūlike ʻana i ka ukana ma ka gel i hoʻoluʻu ʻia) ma 2x SDS loading buffer (Merck, UK) i hoʻoheheʻe ʻia i ʻelua manawa. Ua hoʻokaʻawale ʻia nā protein ma kahi replica 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). Ua hoʻoluʻu ʻia kahi gel me Coomassie Brilliant Blue (Bio-Rad, UK) e hoʻouka a nānā i ka subunit RC-L. Ua hoʻoili ʻia ka protein ma ka lua o ka gel i ka membrane polyvinylidene fluoride (PVDF) i hoʻāla ʻia e ka methanol (Thermo Fisher Scientific, UK) no ka immunoassay. Ua ālai ʻia ka membrane PVDF i loko o 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 a me 5% (w / v) pauka waiū momona, a laila ua hoʻoulu ʻia me ka antibody mua anti-His (ma Dilute the antibody buffer [50 mM tris-HCl (pH 7.6), 150 mM NaCl a me 0.05% (v/v) Tween-20] i loko o 1:1000 A190-114A, Bethyl Laboratories, USA) no 4 mau hola. Ma hope o ka holoi ʻana i 3 mau manawa no 5 mau minuke i loko o ka buffer antibody, ua hui pū ʻia ka membrane me ka horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (diluted 1:10,000 i loko o ka buffer antibody) Incubate e ʻae i ka ʻike ʻana (5 mau minuke ma hope o 3 mau holoi i loko o ka buffer antibody) me ka hoʻohana ʻana i ka WESTAR ETA C 2.0 chemiluminescence substrate (Cyanagen, Italia) a me Amersham Imager 600 (GE Healthcare, UK).
Ma ke kahakiʻi ʻana i ka hoʻolaha ʻana o ka ikaika o kēlā me kēia gel i hoʻoluʻu ʻia a i ʻole ke ala immunoassay, e hoʻohui ana i ka ʻāpana ma lalo o ka piko a me ka helu ʻana i ka lakio ikaika o RC-L (gel i hoʻoluʻu ʻia) a me Protein-W (immunoassay), ma ImageJ (57) Hana i ke kiʻi. Ua hoʻololi ʻia kēia mau lakio i nā lakio molar ma ka manaʻo ʻana he 1:1 ka lakio o RC-L i ka protein-W i loko o ka laʻana RC-LH114-W maʻemaʻe a me ka hoʻomaʻamaʻa ʻana i ka ʻikepili holoʻokoʻa.
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ʻO David JK Swainsbury, ʻo Park Qian, ʻo Philip J. Jackson, ʻo Kaitlyn M. Faries, ʻo Dariusz M. Niedzwiedzki, ʻo Elizabeth C. Martin, ʻo David A. Farmer, ʻo Lorna A. Malone, ʻo Rebecca F. Thompson, ʻo Neil A. Ranson, ʻo Daniel P Canniffe, ʻo Mark J. Dickman, ʻo Dewey Holten, ʻo Christine Kirmaier, ʻo Andrew Hitchcock, ʻo C. Neil Hunter
ʻO ke ʻano hoʻonā kiʻekiʻe o ka paʻakikī light trap 1 i loko o ke kikowaena hopena e hāʻawi i nā ʻike hou i ka dynamics quinone.
ʻO David JK Swainsbury, ʻo Park Qian, ʻo Philip J. Jackson, ʻo Kaitlyn M. Faries, ʻo Dariusz M. Niedzwiedzki, ʻo Elizabeth C. Martin, ʻo David A. Farmer, ʻo Lorna A. Malone, ʻo Rebecca F. Thompson, ʻo Neil A. Ranson, ʻo Daniel P Canniffe, ʻo Mark J. Dickman, ʻo Dewey Holten, ʻo Christine Kirmaier, ʻo Andrew Hitchcock, ʻo C. Neil Hunter
ʻO ke ʻano hoʻonā kiʻekiʻe o ka paʻakikī light trap 1 i loko o ke kikowaena hopena e hāʻawi i nā ʻike hou i ka dynamics quinone.
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