Adilesi yapano: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Malo opangira kuwala otchedwa reaction center light-harvesting complex 1 (RC-LH1) ndiye gawo lalikulu la photosynthetic la mabakiteriya ofiirira otchedwa phototrophic bacteria. Tinayambitsa mapangidwe awiri a cryo-electron microscopy a RC-LH1 complex ochokera ku Rhodopseudomonas palustris. Kapangidwe ka 2.65-Å resolution ka RC-LH114-W complex kali ndi ma subunit 14 a LH1 loops ozungulira RC, omwe amasokonezedwa ndi protein W, pomwe complex yopanda protein-W ndi RC composition yozunguliridwa ndi RC. Ma subunit 16 a LH1 loop otsekedwa. Kuyerekeza kwa mapangidwe awa kumapereka chidziwitso cha mphamvu ya quinone mu RC-LH1 complex, kuphatikizapo kusintha kwa mawonekedwe komwe sikunadziwike kale pomanga quinone pamalo a RC QB, komanso malo a malo othandizira omanga quinone, omwe amathandiza kuwapititsa ku RC. Kapangidwe kapadera ka protein ya W kamaletsa kutsekedwa kwa LH1 loop, motero amapanga njira yofulumizitsira kusinthana kwa quinone/quinolone.
Mphamvu yoperekedwa ndi photosynthesis imatha kuchirikiza pafupifupi zamoyo zonse padziko lapansi, ndipo ili ndi kuthekera kwakukulu kwa sayansi ya zamoyo ya dzuwa. Ngakhale ikulimbikitsa photosynthesis yapadziko lonse, mabakiteriya ofiirira a phototrophic amasonyezanso njira zosiyanasiyana zamagetsi ndi mphamvu zamagetsi. Amatha kupewa photosynthesis ndikukula pamene mabakiteriya a heterotrophic mumdima, amatha kukonza nayitrogeni ndi carbon dioxide, kupanga haidrojeni, ndikuwononga mankhwala a aromatic (1-3). Kuti apereke mphamvu pazinthuzi, kuwala kuyenera kusinthidwa mwachangu komanso moyenera kukhala mphamvu ya mankhwala. Njirayi imayamba pamene antenna complex yolumikizira kuwala imatenga kuwala ndikusamutsa mphamvu yogwidwayo kupita ku reaction center (RC), potero kuyambitsa kulekanitsa mphamvu (4 - 7). Gawo loyambira la photosynthesis mu mabakiteriya ofiirira a phototrophic limapangidwa ndi mtundu wa 2 RC, wozunguliridwa ndi light-harvesting complex 1 (LH1), ndikupanga RC-LH1 core complex. LH1 imapangidwa ndi gulu la αβ heterodimers yokhotakhota, iliyonse yomwe imamanga mabakiteriya awiri a chlorophyll (BChl) mamolekyulu ndi carotenoid imodzi kapena ziwiri (8-12). Antena yosavuta kwambiri ya LH1 imakhala ndi ma heterodimer 16 kapena 17 a αβ omwe amazungulira RC (9-13) mu loop yotsekedwa, koma m'ma complex ena apakati, ma peptide a transmembrane amasokoneza kupitiliza kwa LH1 yozungulira, motero amalimbikitsa kufalikira kwa quinol/quinone pakati pa RC ndi cytochrome bc1 complex (11, 13-15). Chomera chofiirira cha phototrophic Rhodopseudomonas (Rps.) ndi chamoyo choyimira chomwe chingamvetse mphamvu ndi kusamutsa kwa ma elekitironi komwe kumathandizira photosynthesis. Kapangidwe koyamba ka kristalo ka Rps. Chitsanzo cha palustris RC-LH1 complex ndi RC, chozunguliridwa ndi ma loops 15 a heterodimeric LH1, omwe amasokonezedwa ndi puloteni yosadziwika yotchedwa "Protein W" (14). Pambuyo pake Protein-W idazindikirika kuti RPA4402, yomwe ndi puloteni ya 10.5kDa yosadziwika yokhala ndi ma helices atatu a transmembrane (TMH) omwe adanenedweratu (16). Tikupereka lingaliro losintha dzina la puloteni W ya jini ya rpa4402 kukhala pufW kuti igwirizane ndi mayina omwe amagwiritsidwa ntchito pa majini omwe amalemba ma RC-L, M (pufL, pufM) ndi LH1α, β (pufA, pufB) subunits. Chochititsa chidwi n'chakuti, puloteni-W imapezeka pafupifupi 10% ya RC-LH1, zomwe zimasonyeza kuti Rps. palustris imapanga ma complexes awiri osiyana a RC-LH1. Pano, tikupereka lipoti la kapangidwe ka cryo-EM (cryo-EM) ka high-resolution ka ma complexes awiri apakati, imodzi yokhala ndi protein W ndi 14 αβ heterodimers, inayo yopanda protein W ndi closed 16 Heterodimer LH1 loop. Kapangidwe kathu kakuyimira kusintha pang'ono pakumvetsetsa kwa RC-LH1 complex ya Rps. palustris, chifukwa tasanthula kuchuluka kwa anthu ofanana kwa mtundu uliwonse ndipo tili ndi resolution yokwanira yogawa bwino peptide iliyonse ndi ma pigment omangiriridwa ndi ma lipids ndi ma quinones okhudzana nawo. Kuyerekeza kwa kapangidwe kameneka kukuwonetsa kuti mapuloteni atatu a TMH-W omwe sanapezeke mu RC-LH1 complex ina iliyonse mpaka pano amapanga njira ya quinone kuti ifulumizitse kusinthana kwa quinone/quinolone. Malo angapo osungira mafuta ndi quinone omangira apezeka, ndipo tavumbulutsa kusintha kwatsopano pambuyo pa kuphatikiza kwa quinone ndi RC, komwe kungakhale koyenera kwa photosystem II (PSII) RC ya zamoyo zokhala ndi mpweya wa phototrophic. Zomwe tapeza zikupereka chidziwitso chatsopano pa kinetics ya kumanga ndi kusinthana kwa quinone/quinolone mu RC-LH1 core complex ya mabakiteriya ofiirira a phototrophic.
Pofuna kuthandizira kafukufuku watsatanetsatane wa ma complexes awiri omwe amapezeka mu Rps. palustris, timasiyanitsa RC-LH1 iliyonse pogwiritsa ntchito njira za biochemical. Puloteni W-deficient complex (yomwe pano imatchedwa ΔpufW) idayeretsedwa kuchokera ku strain yomwe ilibe jini la pufW (16), ndipo RC-LH1 complex imodzi yokha ndi yomwe ingapangidwe. Puloteni W-containing complex imapangidwa ndi strain. Puloteni W ya strain iyi imasinthidwa ndi chizindikiro cha His cha 10x pa C-terminus yake, kotero kuti protein W-containing complex ikhoza kuphatikizidwa bwino ndi protein W yambiri yomwe ilibe poletsa chitsulo. Chomerachi chimalekanitsidwa bwino (16) Affinity Chromatography (IMAC).
Monga momwe zasonyezedwera pa Chithunzi 1, ma complex onsewa ali ndi ma sub-unit atatu a RC (RC-L, RC-M ndi RC-H) ozunguliridwa ndi antenna ya LH1. Kapangidwe ka 2.80-A ka complex yopanda protein-W kamasonyeza ma heterodimer 16 a αβ, omwe amapanga lupu yotsekedwa ya LH1 yozungulira RC, yomwe imatchedwa RC-LH116 complex. Kapangidwe ka 2.65Å ka complex yokhala ndi protein-W kali ndi 14-heterodimer LH1 yosokonezedwa ndi protein-W, yomwe imatchedwa RC-LH114-W.
(A ndi B) Kuyimira pamwamba pa chinthucho. (C ndi D) Ma pigment ogwirizana omwe amawonetsedwa mu ndodo. (E ndi F) Ma complex omwe amawonedwa kuchokera pamwamba pa cytoplasmic ali ndi ma peptides ndi ma subunits a LH1 omwe amawonetsedwa mu zojambula, ndipo amawerengedwa mozungulira kuchokera ku protein-W gap [yogwirizana ndi Rba numbering. sphaeroides complex (13)]. Pa LH1-α, mtundu wa protein subunit ndi wachikasu; pa LH1-β, mtundu wa protein subunit ndi wabuluu; pa protein-W, protein ndi wofiira; pa RC-H, ndi cyan; pa RC-L, ndi lalanje; pa RC-M, magenta. Ma Cofactors amaimiridwa ndi ndodo, zobiriwira zimayimira BChl ndi BPh mamolekyulu, zofiirira zimayimira carotenoids, ndipo zachikasu zimayimira mamolekyulu a UQ10. (G ndi H) Mawonekedwe okulira a protein-W gap m'dera lofanana la RC-LH114-W complex (G) ndi RC-LH116 complex (H). Ma Cofactor amawonetsedwa ngati malo odzaza, quinone yodulidwa imawonetsedwa ngati buluu. Mpata wa protein-W umawonetsedwa ndi mzere wabuluu wodulidwa mu (G), ndipo mabowo ang'onoang'ono omwe quinone/quinolol imafalikira pa mphete ya LH116 amawonetsedwa ndi mzere wakuda wodulidwa mu (H).
Chithunzi 1 (A ndi B) chikuwonetsa RC yozunguliridwa ndi ma arrays otseguka kapena otsekedwa a LH1αβ heterodimers, iliyonse yomwe imalumikiza BChl awiri ndi carotenoid imodzi (Chithunzi 1, C ndi D). Kafukufuku wakale wasonyeza kuti Rps ndiye LH1 complex. Mu njira yopangira biosynthesis ya spirulina xanthin, mitundu iyi ili ndi mitundu yosiyanasiyana ya carotenoids (17). Komabe, spiropyrroxanthin ndiye carotenoid yayikulu ndipo kuchuluka kwake ndikokwanira. Chifukwa chake, tinasankha kupanga chitsanzo cha spiroxanthin pamalo onse omangira a LH1. Ma polypeptides a alpha ndi beta ndi ma TMH amodzi okhala ndi madera akunja afupiafupi (Chithunzi 1, A, B, E, ndi F). Ngakhale kuchuluka kwa zotsalira 17 pa C-terminus sikunawonekere, alpha polypeptide idadulidwa kuchokera ku Met1 kupita ku Ala46 m'ma complexes onse awiri. β polypeptide inachepetsedwa kuchoka pa Gly4 kufika pa Tyr52 mu RC-LH116, ndipo kuchokera pa Ser5 kufika pa Tyr52 mu RC-LH114-W. Palibe kuchuluka kwa ma residues a N-terminal 3 kapena 4 kapena 13 C-terminal omwe adawonedwa (Chithunzi S1). Kusanthula kwa Mass spectrometry kwa mixed RC-LH1 complex yokonzedwa kuchokera ku wild-type strain kunawonetsa kuti dera lomwe silinapezeke linali chifukwa cha kugawanika kwa ma peptide awa (Chithunzi S1 ndi S2). Fomula ya N-terminal ya α-Met1 inawonedwanso (f). Kusanthula kunawonetsa kuti α-peptide ili ndi ma residues fMet1 kupita ku Asp42/Ala46/Ala47/Ala50, ndipo β-peptide ili ndi ma residues Ser2 kupita ku Ala53, zomwe zikugwirizana bwino ndi mapu a low-temperature EM density.
Kugwirizana kwa α-His29 ndi β-His36 kumapangitsa BChs kukhala maso ndi maso; heterodimer iliyonse ya αβ imasonkhana ndi oyandikana nawo kuti apange loop yotseguka (RC-LH114-W) kapena loop yotsekedwa (RC-LH116) mozungulira RC. Mtundu wa pigment wolumikizidwa ndi exciton (Chithunzi 1, C ndi D). Poyerekeza ndi gulu la 877 nm la RC-LH114-W, kusintha kofiira kwa 880 nm kwa RC-LH116 ndi 3 nm (Chithunzi 2A). Komabe, circular dichroism spectrum ndi yofanana (Chithunzi 2B), zomwe zikusonyeza kuti ngakhale pali kusiyana komveka pakati pa loop yotseguka ndi yotsekedwa, malo am'deralo a BChs ndi ofanana kwambiri. Kusinthasintha kofiira kwa absorption kungakhale chifukwa cha kuchepa kwa kayendedwe ka kutentha ndi kukhazikika kwakukulu pa loop yotsekedwa (18, 19), kusintha kwa kuphatikiza kwa pigment komwe kumachitika chifukwa cha loop yotsekedwa (20, 21), kapena kuphatikiza kwa zotsatira ziwirizi (11).
(A) Ultraviolet/visible/near-infrared absorption spectrum, zomwe nsonga zake zimalembedwa ndi utoto wofanana ndipo zimasinthidwa kukhala BPh peak pa 775 nm. (B) Circular dichroism spectrum imasinthidwa kukhala BChl absorbance pa 805 nm. (C ndi D) Ma ΔA spectra osankhidwa kuchokera ku nthawi yokhazikika ya RC-LH114-W complex (C) ndi RC-LH116 complex (D). Kuti zigwirizane bwino, ma spectra onse amasinthidwa kukhala ∆A ya −A pa 0.2 ps. (E) Mlingo wa cytochrome c2 oxidation pambuyo pa kuwala pamaso pa kuchuluka kosiyanasiyana kwa UQ2 (onani Chithunzi S8 kuti mudziwe zambiri). (F) M'maselo omwe amakula pansi pa kuwala kochepa, kwapakatikati kapena kwamphamvu kwambiri (10, 30 kapena 300μMm-2 s-1, motsatana), mapuloteni W ndi RC-L amagawana mu complex yoyeretsedwa ndi chiŵerengero cholekanitsidwa cha membrane. Dziwani kuchuluka kwa mapuloteni pogwiritsa ntchito SDS-polyacrylamide gel electrophoresis ndi immunoassay (onani Chithunzi S9 kuti mudziwe zambiri). Dziwani kuchuluka kwa mapuloteni poyerekeza ndi RC-LH114-W complex yoyeretsedwa. Chiŵerengero cha stoichiometric cha RC-L ku protein-W ya complex ndi 1:1.
Ma BChl omwe ali pamalo 1 mu loop yolakwika ya αβ14 ya RC-LH114-W (Chithunzi 1, A, C, ndi E) ali pafupi ndi RC primary donor (P) ndi 6.8Å kuposa ma BChl ofanana mu RC-LH116 (Chithunzi 1, B, D, ndi F, ndi Chithunzi S3); komabe, kinetics ya absorption ya transient ya ma complexes awiriwa ikuwonetsa kuti pa RC-LH114-W ndi RC-LH116, ma constants a excitation energy transfer time constants kuchokera ku LH1 kupita ku RC ndi 40 ±4 ndi 44±3 ps (Chithunzi 2). , C ndi D, Chithunzi S4 ndi Table S2). Palibenso kusiyana kwakukulu pakusamutsa kwamagetsi mkati mwa RC (Chithunzi S5 ndi zolemba zina zowonjezera). Tikuganiza kuti kulumikizana kwapafupi kwa nthawi yosamutsa mphamvu pakati pa LH1 ndi RC-P kumachitika chifukwa cha mtunda wofanana, ngodya ndi mphamvu zomwe zingatheke za BChl zambiri m'ma loops awiri a LH1. Zikuwoneka kuti kufufuza njira ya mphamvu ya LH1 kuti mufike pa mtunda wocheperako sikofulumira kuposa kusamutsa mphamvu mwachindunji kuchokera kumalo osakwanira kupita ku RC. Chizunguliro chotseguka cha LH1 mu RC-LH114-W chingayendenso pang'ono kutentha pansi pa kutentha kochepa kuti chiwunikire kapangidwe kake, ndipo pali mawonekedwe a mphete ya αβ14 yayitali kutentha kwa chipinda kuchokera pa mtunda wa pigmentation wa βBChls pamalo a RC 1.
Gulu la RC-LH116 lili ndi ma BChl 32 ndi ma carotenoid 16, ndipo kapangidwe kake konsekonse ndi kofanana ndi komwe kamapezeka kuchokera ku Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 strain (PDB ID 7C9R) (12) ndi algae wobiriwira (Blc.viridis) (PDB ID 6ET5) (10). Pambuyo pa kulinganiza, kusintha pang'ono kokha m'malo a αβ heterodimers kunawonedwa, makamaka 1-5, 15, ndi 16 (Chithunzi S6). Kupezeka kwa protein-W kumakhudza kwambiri kapangidwe ka LH1. Ma TMH ake atatu amalumikizidwa ndi ma loops afupiafupi, ndi N-terminal kumbali ya lumen ya gululo ndi C-terminal kumbali ya cytoplasmic (Zithunzi 1A ndi 3, A mpaka D). Mapuloteni-W nthawi zambiri amakhala ndi hydrophobic (Chithunzi 3B), ndipo TMH2 ndi TMH3 zimalumikizana ndi LH1αβ-14 kuti apange pamwamba pa membrane (Chithunzi 3, B ndi E mpaka G). Ma interface amapangidwa makamaka ndi zotsalira za Phe, Leu ndi Val m'chigawo cha transmembrane. Zotsalira izi zimadzazidwa ndi ma amino acid a hydrophobic ndi ma pigment a αβ-14. Zotsalira zina za polar zimathandizanso kuyanjana, kuphatikizapo mgwirizano wa hydrogen pakati pa W-Thr68 ndi β-Trp42 pamwamba pa cavity yovuta (Chithunzi 3, F ndi G). Pamwamba pa cytoplasm, Gln34 ili pafupi ndi gulu la keto la αβ-14 carotenoids. Kuphatikiza apo, molekyulu ya n-dodecyl β-d-maltoside (β-DDM) idathetsedwa, ndipo mchira wake wa hydrophobic unakulitsidwa mpaka pakati pa protein-W ndi αβ-14, ndipo mchira wa lipid ukhoza kukhala m'thupi. Tinaonanso kuti madera a C-terminal resolution a protein W ndi RCH ali pafupi kwambiri, koma osati mkati mwa malo opangira kuyanjana kwina (Chithunzi 1, A ndi E). Komabe, pakhoza kukhala kuyanjana mu C-terminal amino acids osasinthidwa a mapuloteni awiriwa, zomwe zingapereke njira yopezera protein-W panthawi yosonkhanitsa RC-LH114-W complex.
(A) Puloteni-W, yomwe imayang'anizana ndi mawonekedwe a LH1αβ14 mu mawonekedwe a katuni, ili ndi unyolo wam'mbali wooneka ngati ndodo (wofiira), womwe umawonetsedwa mu gawo la chithunzi cha electrostatic potential (pamwamba pa imvi yowonekera bwino yokhala ndi mulingo wa contour wa 0.13). (B) Puloteni-W yomwe imayimiridwa ndi malo okhala ndi mtundu wa hydrophobic. Madera okhala ndi polar ndi charged amawonetsedwa mu cyan, madera okhala ndi hydrophobic amawonetsedwa mu zoyera, ndipo madera okhala ndi hydrophobic kwambiri amawonetsedwa mu lalanje. (C ndi D) Puloteni-W yomwe imawonetsedwa mu katuni, mawonekedwe ake ndi ofanana ndi a (A) (C), ndipo amazunguliridwa ndi 180° (D). Malinga ndi malo omwe ali mu sequence, zotsalira zomwe zimasiyanitsa zimatenga mtundu wa utawaleza, pomwe N-terminal ndi yabuluu ndipo C-terminal ndi yofiira. (E) Puloteni-W yomwe ili mu mawonekedwe ofanana ndi a (A), ndipo zotsalira zomwe zili pa mawonekedwe a protein-W:LH1 zimayimiridwa ndi ndodo zokhala ndi zizindikiro zomangiriridwa. (F) Puloteni-W imazunguliridwa 90° poyerekeza ndi (E) ndi LH1αβ14 mu chithunzi cha katuni, komanso poyerekeza ndi zotsalira za mawonekedwe mu chithunzi cha bar. Zotsalira zomwe zili pamwamba pa beta polypeptide zimalembedwa. Cofactor imawonetsedwa ngati bala yofanana ndi mtundu wa Chithunzi 1, β-DDM yowola imawonetsedwa mu imvi, ndipo mpweya umawonetsedwa mu wofiira. (G) Mawonekedwe mu (F) amazunguliridwa 180°, ndi zotsalira zodziwika bwino za alpha polypeptide yolembedwa.
Puloteni-W imalowa m'malo mwa αβ heterodimer (ya 15 mu Chithunzi 1F), motero imaletsa kutseka kwa loop ndikupotoza ma heterodimer atatu oyamba a αβ. Zinaonedwa kuti ngodya yayikulu kwambiri ya heterodimer yoyamba ya αβ-1 poyerekeza ndi filimu yachibadwa inali 25° mpaka 29° (Chithunzi 1, A ndi E), yomwe idapangidwa ndi kupendekera kwa 2° mpaka 8° kwa αβ-1 mu RC A sharp contrast-LH116 (Chithunzi 1, B ndi F). Ma heterodimer achiwiri ndi achitatu amapendekeka pa 12° mpaka 22° ndi 5° mpaka 10°, motsatana. Chifukwa cha choletsa cha steric cha RC, kupendekeka kwa αβ-1 sikuphatikizapo awiri achiwiri a αβ (omwe akufanana ndi 16th αβ mu Chithunzi 1F), motero amapanga mpata wowonekera mu mphete ya LH1 (Chithunzi 1, A ndi E). Chifukwa cha kusowa kwa ma heterodimer awiri a αβ, limodzi ndi kutayika kwa ma BChl anayi ndi ma carotenoid awiri, palibe ma carotenoid omwe amamangirira ku subunit yopotoka ya αβ-1, zomwe zimapangitsa kuti pakhale mphete ya LH114-W yokhala ndi ma carotenoid 13 a Vegetarian ndi ma BChl 28. Ziwerengero za malo okhala ma complexes awiriwa m'madera a αβ1 mpaka 7 ndizochepa kuposa za ena onse a LH1 loop, zomwe zingasonyeze kupangika kwa pulasitiki kwa subunit ya LH1 pafupi ndi malo a RC QB (Chithunzi 4).
Zithunzi za RC-LH114-W (A ndi B) ndi RC-LH116 (C ndi D) zikuwonetsedwa kuchokera pamwamba/mbali yomweyo (A ndi B) (A ndi C) ndi pamwamba pa mpata wa Chithunzi 1. (B ndi D). Makiyi amitundu akuwonetsedwa kumanja.
Chinthu china chokhacho cha core complex chomwe chili ndi stoichiometric ratio ya 1:14 ndi Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Komabe, protein W ndi PufX zilibe homology yodziwikiratu, ndipo zimakhudza kwambiri kapangidwe kawo ka LH1. PufX ndi TMH imodzi yokhala ndi N-terminal cytoplasmic domain yomwe imagwirizana ndi mbali ya cytoplasmic ya RC-H subunit (13) pamalo ofanana ndi Rps. palustris LH116αβ-16. PufX imapanga njira yosinthira quinone/quinolone pakati pa RC-LH1 ndi cytochrome bcl complex ndipo imapezeka mu Rba. sphaeroides core complex yonse (13). Ngakhale kuti monomer-monomer interface ili mu Rba. Dimer ya sphaeroides RC-LH1-PufX ili pamalo omangirira a protein W mu RC-LH114-W, ndipo kusiyana komwe kumabwera chifukwa cha PufX ndi protein-W kuli pamalo ofanana (Chithunzi S7A). Kusiyana kwa RC-LH114-W kumagwirizananso ndi njira ya quinone (8) ya Pseudomonas rosea LH1, yomwe imapangidwa ndi ma peptides osakhudzana ndi protein W kapena PufX (Chithunzi S7B). Kuphatikiza apo, njira ya quinone mu Blc. LH1 yobiriwira ya emerald yopangidwa pochotsa subunit imodzi ya γ (7) ili pamalo ofanana (Chithunzi S7C). Ngakhale kuti imayendetsedwa ndi mapuloteni osiyanasiyana, mawonekedwe a njira za quinone/quinolol izi pamalo ofanana mu RC-LH1 complex akuwoneka ngati chitsanzo cha kusintha kogwirizana, kusonyeza kuti kusiyana komwe kumabwera chifukwa cha protein W kumatha kugwira ntchito ngati njira ya quinone.
Mpata womwe uli mu LH114-W loop umalola kupangidwa kwa dera la nembanemba lopitirira pakati pa malo amkati a RC-LH114-W complex ndi nembanemba yochuluka (Chithunzi 1G), m'malo molumikiza madera awiriwa kudzera mu puloteni monga momwe zilili m'mapuloteni. RC-LH116 complex ndi yofanana ndi Tch. Needle-like complex yotsekedwa (22) (Chithunzi 1H). Popeza kufalikira kwa quinone kudzera mu nembanemba kumathamanga kuposa kufalikira kudzera mu njira yopapatiza ya protein, loop yotseguka ya LH114-W ingalole kusintha kwa RC mwachangu kuposa loop yotsekedwa ya LH116, ndipo kufalikira kwa quinone mu RC kungakhale kocheperako. Pofuna kuyesa ngati puloteni W imakhudza kusintha kwa quinones kudzera mu RC, tinachita mayeso a cytochrome oxidation pa kuchuluka kwina kwa ubiquinone 2 (UQ2) (analogue ya UQ10 yachilengedwe yokhala ndi mchira waufupi wa isoprene) (Chithunzi 2E). Ngakhale kukhalapo kwa chelated quinone kumalepheretsa kutsimikiza molondola kwa Michaelis constant (RC-LH114-W ndi RC-LH116 ndizoyenera 0.2±0.1μM ndi 0.5±0.2μM, motsatana), kuchuluka kwakukulu kwa RC-LH114-W (4.6±0.2 e-RC-1 s-1) ndi 28±5% kuposa RC-LH116 (3.6±0.1 e-RC-1 s-1).
Poyamba tinayerekeza kuti puloteni-W ilipo pafupifupi 10% ya core complex (16); apa, kuchuluka kwa maselo okulirapo omwe ali ndi kuwala kochepa, pakati, ndi kuwala kwakukulu ndi 15±0.6%, 11±1% ndi 0.9±0.5, motsatana % (Chithunzi 2F). Kuyerekeza kochuluka kwa mass spectrometry kunawonetsa kuti kuwonjezera kwa histidine tag sikunachepetse kuchuluka kwa puloteni-W poyerekeza ndi mitundu yachilengedwe (P = 0.59), kotero milingo iyi si chinthu chopangidwa ndi puloteni-W yosinthidwa (Chithunzi S10). Komabe, kuchepa kwa puloteni-W mu RC-LH1 complex kungalole ma RC ena kusintha mofulumira, motero kuchepetsa kusinthana kwa quinone/quinolone pang'onopang'ono mu RC-LH116 complex. Tinaona kuti kuchuluka kwa kuwala kwakukulu sikukugwirizana ndi deta yaposachedwa ya transcriptomics, zomwe zikusonyeza kuti mawonekedwe a majini a pufW amawonjezeka pansi pa kuwala kwamphamvu (Chithunzi S11) (23). Kusiyana pakati pa kulembedwa kwa pufW ndi kulowetsedwa kwa protein-W mu RC-LH1 complex n'kosokoneza ndipo kungasonyeze momwe puloteniyi imagwirira ntchito movutikira.
Mu RC-LH114-W, mamolekyu 6 a cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) ndi 29 β-DDM amagawidwa ndikupangidwa momwemo ma CDL 6, 24 POPC, 2 POPG ndi 12 βDDM. RC-LH116 (Chithunzi 5, A ndi B). Mu kapangidwe kake kawiri, CDL ili pafupi kukhala mbali ya cytoplasmic ya complex, pomwe POPC, POPG ndi β-DDM nthawi zambiri zimakhala mbali ya luminal. Mamolekyu awiri a lipid ndi detergent adalekanitsidwa m'chigawo cha αβ-1 mpaka αβ-6 cha RC-LH114-W complex (Chithunzi 5A), ndipo asanu adalekanitsidwa m'chigawo chofanana cha RC-LH116 (Chithunzi 5B). Ma lipids ambiri adapezeka mbali ina ya complex, makamaka CDL, omwe adasonkhanitsidwa pakati pa RC ndi αβ-7 mpaka αβ-13 (Chithunzi 5, A ndi B). Ma lipids ena okonzedwa bwino ndi zotsukira ali kunja kwa mphete ya LH1, ndipo maunyolo a acyl okonzedwa bwino amafalikira pakati pa ma subunits a LH1, omwe amadziwika kuti β-DDM mu RC-LH114-W, ndipo amatanthauzidwa kuti β-DDM mu RC A osakaniza a β-DDM ndi POPC-LH116. Malo ofanana a chelating lipids ndi zotsukira mu kapangidwe kathu akuwonetsa kuti ndi malo omangira ogwirizana ndi thupi (Chithunzi S12A). Malo a mamolekyu ofanana mu Tch alinso ndi kusinthasintha kwabwino. Wofatsa komanso Trv. Strain 970 RC-LH1s (Chithunzi S12, B mpaka E) (9, 12) ndi zotsalira za hydrogen-bonding za gulu la lipid head zinasonyeza kusungidwa bwino mu sequence alignment (Chithunzi S13), zomwe zikusonyeza kuti Conserved CDL yomwe imagwirizana ndi RC (24), malo awa akhoza kusungidwa mu RC-LH1 complex.
(A ndi B) Ma peptide a RC-LH114-W (A) ndi RC-LH116 (B) amaimiridwa ndi zojambula, ndipo utoto umaimiridwa ndi ndodo, pogwiritsa ntchito mtundu womwe uli mu Chithunzi 1. Ma lipid akuwonetsedwa mu utoto wofiira, ndipo sopo wothira madzi akuwonetsedwa mu imvi. UQ yolumikizidwa ku malo a RC QA ndi QB ndi yachikasu, pomwe UQ yodzipatula ndi yabuluu. (C ndi D) Mawonekedwe ofanana ndi (A) ndi (B), ndi mafuta osiyidwa. (E mpaka G) Mawonekedwe okulirapo a Q1(E), Q2(F) ndi Q3(G) kuchokera ku RC-LH116, ndi maunyolo am'mbali omwe amakhudza wina ndi mnzake. Ma hydrogen bond akuwonetsedwa ngati mizere yakuda yodulidwa.
Mu RC-LH116, RC QA ndi QB UQ zonse, zomwe zimatenga nawo mbali mu kusamutsa ma elekitironi mu njira yolekanitsa mphamvu, zimawonongeka m'malo awo omangira. Komabe, mu RC-LH114-W, QB quinone sinathetsedwe ndipo tidzakambirana mwatsatanetsatane pansipa. Kuwonjezera pa QA ndi QB quinones, mamolekyu awiri a UQ odulidwa (omwe ali pakati pa mphete za RC ndi LH1) amagawidwa mu kapangidwe ka RC-LH114-W malinga ndi magulu awo amutu okonzedwa bwino (omwe ali mu Q1 ndi Q2, motsatana). Chithunzi 5C). Mayunitsi awiri a isoprene aperekedwa ku Q1, ndipo mapu a density amathetsa michira yonse 10 ya isoprene ya Q2. Mu kapangidwe ka RC-LH16, mamolekyu atatu a UQ10 odulidwa (Q1 mpaka Q3, Chithunzi 5D) adathetsedwera, ndipo mamolekyu onse ali ndi density yomveka bwino mchira wonse (Chithunzi 5, D mpaka G). Mu kapangidwe kake kawiri, malo a magulu a mutu wa quinone a Q1 ndi Q2 ali ndi kusinthasintha kwabwino (Chithunzi S12F), ndipo amangogwirizana ndi RC. Q1 ili pakhomo la W gap ya RC-LH114-W (Chithunzi 1G ndi 5, C, D ndi E), ndipo Q2 ili pafupi ndi malo omangira QB (Chithunzi 5, C, D) ndi F). Zotsalira za L-Trp143 ndi L-Trp269 zosungidwa zili pafupi kwambiri ndi Q1 ndi Q2 ndipo zimapereka kuyanjana kwa π-stacking (Chithunzi 5, E ndi F, ndi Chithunzi S12). L-Gln88, 3.0 Å kuchokera ku distal oxygen ya Q1, imapereka mgwirizano wamphamvu wa hydrogen (Chithunzi 5E); zotsalira izi zimasungidwa mu ma RC onse kupatula ubale wakutali kwambiri (Chithunzi S13). L-Ser91 imalowetsedwa m'malo mwa Thr m'ma RC ena ambiri (Chithunzi S13), ndi ma Angstroms 3.8 ochokera ku methyl oxygen ya Q1, ndipo ingapereke ma hydrogen bond ofooka (Chithunzi 5E). Q3 ikuwoneka kuti ilibe mgwirizano wapadera, koma ili m'dera la hydrophobic pakati pa RC-M subunit ndi LH1-α subunit 5 mpaka 6 (Chithunzi 5, D ndi G). Q1, Q2 ndi Q3 kapena ma quinones apafupi omwe ali ndi chelated nawonso atha kuthetsedwa mu Tch. Gentle, Trv. Strain 970 ndi Blc. Kapangidwe ka iris (9, 10, 12) kamasonyeza malo osungira othandizira a quinone mu RC-LH1 complex (Chithunzi S12G). Ma UQ asanu owonongeka mu RC-LH116 akugwirizana bwino ndi 5.8±0.7 ya complex iliyonse yomwe imatsimikiziridwa ndi high performance liquid chromatography (HPLC), pomwe ma UQ atatu owonongeka mu RC-LH114-W ndi otsika kuposa Mtengo woyezedwa wa 6.2±0.3 (Chithunzi S14) ukuwonetsa kuti pali mamolekyu a UQ osasinthidwa mu kapangidwe kake.
Ma polypeptide a L ndi M omwe ali ndi ma TMH asanu aliwonse amapanga heterodimer yomwe imaphatikiza BChl dimer imodzi, ma BChl monomers awiri, ma bacteriophage awiri (BPh), ndi chitsulo chimodzi chosakhala cha Heme ndi mamolekyulu a UQ10 amodzi kapena awiri. Kudzera mu kukhalapo kwa ma hydrogen bond pa gulu la terminal ketone ndi kusonkhana kwake kodziwika mu Rps, ma carotenoid amaphatikizidwa mu M-subunit, yomwe imatchedwa cis-3,4-dehydroorhodopin. Mitundu (25). Malo akunja a membrane a RC-H amamangiriridwa ku membrane ndi TMH imodzi. Kapangidwe ka RC konse kali kofanana ndi ma subunit atatu a RC a mitundu yofananira (monga Rba). sphaeroides (PDB ID: 3I4D). Ma macrocycles a BChl ndi BPh, msana wa carotenoid ndi chitsulo chosakhala cha heme zimalumikizana mkati mwa resolution range ya kapangidwe kameneka, monganso gulu la mutu wa UQ10 pamalo a QA ndi QB quinone pa RC-LH116 (Chithunzi S15).
Kupezeka kwa mapangidwe awiri a RC okhala ndi kuchuluka kosiyana kwa anthu okhala pamalo a QB kumapereka mwayi watsopano wofufuza kusintha kosasintha komwe kumayenderana ndi kumangidwa kwa QB quinone. Mu RC-LH116 complex, QB quinone ili pamalo omangidwa kwathunthu a "proximal" (26), koma kulekanitsidwa kwa RC-LH114-W kulibe quinone ya QB. Palibe quinone ya QB mu RC-LH114-W, zomwe ndizodabwitsa chifukwa complex imagwira ntchito, kuposa RC-LH116 complex yokhala ndi quinone ya QB yokhazikika. Ngakhale kuti mphete ziwiri za LH1 zimakhala ndi ma quinone pafupifupi asanu ndi limodzi, asanu amakhazikika mwadongosolo mu mphete yotsekedwa ya RC-LH116, pomwe atatu okha ndi ochepa mwadongosolo mu mphete yotseguka ya RC-LH114-W. Kuwonjezeka kwa structural disorder kumeneku kungasonyeze kusintha kwachangu kwa malo a RC-LH114-W QB, kuthamanga kwa quinone kinetics mu complex, komanso kuwonjezeka kwa mwayi wodutsa LH1 loop. Tikuganiza kuti kusowa kwa UQ pamalo a RC QB a RC-LH114-W kungakhale chifukwa cha zovuta komanso zogwira ntchito kwambiri, ndipo malo a QB a RC-LH114-W adayimitsidwa nthawi yomweyo mu kusintha kwa UQ. Gawo lenilenilo (lolowera pamalo a QB latsekedwa) likuwonetsa mawonekedwe a ntchitoyi.
Popanda QB, kuzungulira kwa L-Phe217 komwe kumayenderana ndi malo omwe sagwirizana ndi UQ10 binding, chifukwa kudzayambitsa kugundana kwa malo ndi gawo loyamba la isoprene la mchira (Chithunzi 6A). Kuphatikiza apo, kusintha kwakukulu koonekeratu kwa conformational ndikodziwikiratu, makamaka helix de (helix yaifupi yomwe ili pakati pa TMH D ndi E) komwe L-Phe217 imasamutsidwira ku thumba lomangira la QB ndi kuzungulira kwa L-Tyr223 (Chithunzi 6A) Kuti aswe mgwirizano wa hydrogen ndi chimango cha M-Asp45 ndikutseka khomo la malo omangira a QB (Chithunzi 6B). Helix de pivots pansi pake, Cα ya L-Ser209 imasamutsidwira ndi 0.33Å, pomwe L-Val221Cα imasamutsidwira ndi 3.52Å. Palibe kusintha komwe kungawonekere mu TMH D ndi E, komwe kuli kopanda mphamvu m'mapangidwe onse awiri (Chithunzi 6A). Monga momwe tikudziwira, iyi ndi kapangidwe koyamba mu RC yachilengedwe komwe kamatseka malo a QB. Kuyerekeza ndi kapangidwe kathunthu (komangidwa ndi QB) kukuwonetsa kuti quinone isanachepe, kusintha kwa mawonekedwe kumafunika kuti ilowe mu quinone. L-Phe217 imazungulira kuti ipange kuyanjana kwa π-stacking ndi gulu la mutu wa quinone, ndipo helix imasunthira kunja, kulola mafupa a L-Gly222 ndi unyolo wa mbali wa L-Tyr223 kupanga netiweki ya hydrogen bond yokhala ndi kapangidwe kokhazikika ka hydrogen bond (Chithunzi 6, A ndi C).
(A) Katuni yolumikizana ya hologram (unyolo wa L, unyolo wa lalanje/M, magenta) ndi apo (imvi), momwe ma key residues amawonetsedwa ngati choyimira ngati ndodo. UQ10 imayimiridwa ndi bala lachikasu. Mzere wokhala ndi madontho umasonyeza ma hydrogen bond omwe amapangidwa mu kapangidwe konse. (B ndi C) Kuyimira pamwamba pa apolipoprotein ndi kapangidwe konse ka mphete, kuwonetsa mpweya wa L-Phe217 mu buluu ndi L-Tyr223 mu wofiira, motsatana. L subunit ndi lalanje; ma subunit a M ndi H si amitundu. (D ndi E) Apolipoprotein (D) ndi malo onse a RC QB [mtundu ndi (A) motsatana] ndi Thermophilus thermophilus PSII (wobiriwira, wabuluu wokhala ndi quinone yapulasitiki; PDB ID: 3WU2) Align (58).
Mosayembekezereka, ngakhale kuti pali mapangidwe angapo a ma RC osowa QB opanda LH1, kusintha kwa mawonekedwe komwe kwawonedwa mu kafukufukuyu sikunanenedwepo kale. Izi zikuphatikizapo kapangidwe ka QB kochokera ku Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) ndi Rba. sphaeroides (PDB ID: 1OGV) (29), zomwe zonse zili zofanana ndi kapangidwe kawo konse ka QB. Kuyang'anitsitsa kwa 3PRC kwawonetsa kuti mamolekyulu a sopo a LDAO (Lauryl Dimethyl Amine Oxide) amamangirira pakhomo la malo a QB, zomwe zingalepheretse kukonzedwanso kukhala mawonekedwe otsekedwa. Ngakhale LDAO siiwola pamalo omwewo mu 1EYS kapena 1OGV, ma RC awa amakonzedwa pogwiritsa ntchito sopo yemweyo ndipo chifukwa chake amatha kupanga zotsatira zomwezo. Kapangidwe ka kristalo ka Rba. Sphaeroides RC yolumikizidwa ndi cytochrome c2 (PDB ID: 1L9B) ikuwonekanso kuti ili ndi malo otsekedwa a QB. Komabe, pankhaniyi, dera la N-terminal la RC-M polypeptide (lolumikizana ndi malo omangira a QB kudzera mu H bond ya Tyr residue pa Q helix) limatenga mawonekedwe osazolowereka, ndipo kusintha kwa QB sikunafufuzidwe kwambiri (30). Cholimbikitsa ndichakuti sitinawone mtundu uwu wa kusintha kwa M polypeptide mu kapangidwe ka RC-LH114-W, komwe kuli kofanana ndi dera la N-terminal la RC-LH116 RC. Tiyeneranso kudziwa kuti pambuyo pochotsa antenna ya LH1 yochokera ku detergent, ma apolipoprotein RCs mu PDB adathetsedwa, zomwe zidachotsa ma quinone pools amkati ndi lipids pakati pa RC ndi pamwamba pamkati mwa mphete yozungulira ya LH1 (31, 32). RC imakhalabe yogwira ntchito chifukwa imasunga ma cofactor onse, kupatula QB quinone yomwe imatha kusungunuka, yomwe siikhazikika bwino ndipo nthawi zambiri imatayika panthawi yokonzekera (33). Kuphatikiza apo, zimadziwika kuti kuchotsedwa kwa LH1 ndi mafuta achilengedwe ochokera ku RC kumatha kukhudza ntchito, monga kufupikitsa nthawi ya moyo wa P+QB-state yolekanitsidwa ndi charge (31, 34, 35). Chifukwa chake, tikuganiza kuti kukhalapo kwa mphete ya LH1 yozungulira RC kungapangitse kuti malo a QB "otsekedwa" asungidwe, motero kusunga malo am'deralo pafupi ndi QB.
Ngakhale apolipoprotein (yopanda QB quinone) ndi kapangidwe kake konse zikuyimira zithunzi ziwiri zokha za kusintha kwa malo a QB, osati zochitika zingapo, pali zizindikiro zoti kumangirirako kumatha kutsekedwa kuti kupewe kumangiriranso ndi hydroquinone Kuletsa kuletsa kwa substrate. Kuyanjana kwa quinolol ndi quinone pafupi ndi malo a QB a apolipoprotein kungakhale kosiyana, zomwe zimapangitsa kuti RC ikakane. Kwakhala kunenedwa kwa nthawi yayitali kuti kusintha kwa mawonekedwe kumachita gawo pakumangirira ndi kuchepetsa ma quinone. Kuthekera kwa ma RC ozizira kuchepetsa ma quinones pambuyo poti kusintha kwamdima kwachepa (36); X-ray crystallography ikuwonetsa kuti kuwonongeka kumeneku kumachitika chifukwa cha ma quinone a QB omwe agwidwa mu mawonekedwe a "distal" pafupifupi 4.5 Å kuchokera pamalo ogwirira ntchito a proximal (26), 37). Tikuwonetsa kuti mawonekedwe omangirirako a distal awa ndi chithunzi cha mkhalidwe wapakati pakati pa apolipoprotein ndi kapangidwe ka mphete yonse, komwe kumatsatira kuyanjana koyamba ndi quinone ndi kutsegulidwa kwa malo a QB.
Mtundu wa II RC womwe umapezeka mu PSII complex ya mabakiteriya ena a phototrophic ndi cyanobacteria, algae ndi zomera uli ndi kusungidwa kwa kapangidwe kake ndi ntchito (38). Kulinganiza kwa kapangidwe kake komwe kwawonetsedwa pa Chithunzi 6 (D ndi E) kukugogomezera kufanana pakati pa PSII RCs ndi malo a QB a bacterial RC complex. Kuyerekeza kumeneku kwakhala chitsanzo chophunzirira machitidwe ogwirizana kwambiri a quinone binding ndi reduction. Mabuku am'mbuyomu adanenanso kuti kusintha kwa kapangidwe kake kumayenderana ndi kuchepetsa kwa PSII kwa quinones (39, 40). Chifukwa chake, poganizira za kusungidwa kwa RC, njira yomangira iyi yomwe sinawonedwepo kale ingagwiritsidwenso ntchito pamalo a QB a PSII RC mu zomera za phototrophic zokhala ndi mpweya.
Rps ΔpufW (kuchotsedwa kwa pufW kopanda zilembo) ndi PufW-His (C-terminal 10x His-taged protein-W yochokera ku mitundu yachilengedwe ya pufW locus). palustris CGA009 idafotokozedwa m'ntchito yathu yapitayi (16). Mitundu iyi ndi isogenic wild-type parent idapezeka mufiriji mwa kuyika maselo ochepa pa PYE (5 g iliyonse ya lita -1) (yosungidwa mu LB pa -80 °C, yokhala ndi 50% ( w/v) glycerol) protein, yisiti extract ndi succinate) agar [1.5% ( w/v)] plate. Mbaleyi idayikidwa mu mdima usiku wonse kutentha kwa chipinda pansi pa mikhalidwe ya anaerobic, kenako idawunikiridwa ndi kuwala koyera (~50 μmolm-2 s-1) koperekedwa ndi OSRAM 116-W halogen bulbs (RS Components, UK) kwa masiku 3 mpaka 5 mpaka koloni imodzi itawonekera. Gulu limodzi la koloni linagwiritsidwa ntchito kubaya 10 ml ya M22+ medium (41) yowonjezerapo 0.1% (w/v) casamino acids (pano akutchedwa M22). Chikhalidwechi chinakulitsidwa pansi pa mpweya wochepa mumdima pa 34°C ndikugwedezeka pa 180 rpm kwa maola 48, kenako 70 ml ya chikhalidwecho inayikidwa pansi pa mikhalidwe yomweyi kwa maola 24. Chikhalidwe cha semi-aerobic chokhala ndi voliyumu ya 1 ml chimagwiritsidwa ntchito kubaya 30 ml ya M22 medium mu botolo lagalasi lowonekera la 30 ml lonse ndikuwotchedwa ndi agitation (~50μmolm-2 s-1) kwa maola 48 ndi mphamvu ya magnetic sterile magnetic Stirring rod. Kenako 30 ml ya chikhalidwecho inayikidwa ndi pafupifupi lita imodzi ya chikhalidwe pansi pa mikhalidwe yomweyi, yomwe kenako inagwiritsidwa ntchito kubaya pafupifupi malita 9 a chikhalidwecho omwe amayatsidwa pa ~200 μmolm-2 s-1 kwa maola 72. Maselowo anakololedwa pogwiritsa ntchito centrifugation pa 7132 RCF kwa mphindi 30, kenako anaikidwanso mu ~10 ml ya 20 mM tris-HCl (pH 8.0), ndipo anasungidwa pa -20°C mpaka pakufunika.
Mukasungunuka, onjezani makhiristo ena a deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) ndi mapiritsi awiri a Roche holoenzyme protease inhibitor (Merck, UK) ku maselo omwe adabwezeretsedwanso. Mu selo la 20,000 psi French pressure (Aminco, USA), maselowo adasokonekera maulendo 8 mpaka 12. Pambuyo pochotsa maselo osasweka ndi zinyalala zosasungunuka pogawa 18,500 RCF kwa mphindi 15 pa 4°C, nembanembayo idachotsedwa ku lysate yopaka utoto pogawa 113,000 RCF kwa maola awiri pa 43,000°C. Tayani gawo losungunuka ndikugawanso nembanemba yofiirira mu 100 mpaka 200 ml ya 20 mM tris-HCl (pH 8.0) ndikuyiyikanso mpaka palibe ma aggregates owoneka. Nembanemba yopachikidwayo inayikidwa mu 20 mM tris-HCl (pH 8.0) (Anatrace, USA) yokhala ndi 2% (w/v) β-DDM kwa ola limodzi mumdima pa 4°C ndikusakaniza pang'ono. Kenako centrifuge pa 70°C kuti isungunuke 150,000 RCF pa 4°C kwa ola limodzi kuti ichotse zotsalira zosasungunuka.
Kachidutswa kosungunuka kuchokera ku ΔpufW strain kanagwiritsidwa ntchito pa 50 ml DEAE Sepharose ion exchange column yokhala ndi ma column volumes atatu (CV) a binding buffer [20 mM tris-HCl (pH 8.0) yokhala ndi 0.03% (w / v) β-DDM]. Tsukani column ndi ma CV binding buffers awiri, kenako tsukani column ndi ma binding buffers awiri okhala ndi 50 mM NaCl. RC-LH116 complex inachotsedwa ndi linear gradient ya 150 mpaka 300 mM NaCl (mu binding buffer) pa 1.75 CV, ndipo binding complex yotsalayo inachotsedwa ndi binding buffer yokhala ndi 300 mM NaCl pa 0.5 CV. Sonkhanitsani ma absorption spectrum pakati pa 250 ndi 1000 nm, sungani gawoli ndi absorbance ratio (A880/A280) yoposa 1 pa 880 mpaka 280 nm, lisungunuleni kawiri mu binding buffer, ndikugwiritsanso ntchito njira yomweyi pa DEAE column On purification. Sakanizani magawo ndi ma A880/A280 ratio apamwamba kuposa 1.7 ndi ma A880/A805 ratio apamwamba kuposa 3.0, chitani gawo lachitatu la kusinthana kwa ma ion, ndikusunga magawo ndi ma A880/A280 ratio apamwamba kuposa 2.2 ndi ma A880/A805 ratio apamwamba kuposa 5.0. Chotsukidwa pang'onocho chinayikidwa mu fyuluta ya Amicon 100,000 molecular weight cut-off (MWCO) centrifugal (Merck, UK), ndipo chinayikidwa pa Superdex 200 16/600 size exclusion column (GE Healthcare, US) yokhala ndi 200 mM NaCl buffer, kenako chinachotsedwa mu buffer yomweyo pa 1.5 CV. Sonkhanitsani ma absorption spectra a kukula exclusion fraction, ndikuyika ma absorption spectra ndi ma A880/A280 ratios pa 2.4 ndi A880/A805 ratios pa 5.8 mpaka 100 A880, ndipo nthawi yomweyo mugwiritse ntchito pokonzekera kapena kusungira cryo-TEM grid. Sungani pa -80°C mpaka pakufunika.
Kachidutswa kosungunuka kuchokera ku PufW-His strain kanagwiritsidwa ntchito pa 20 ml HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) yokhala ndi 200 mM NaCl ndi 0.03% (w/w)) mu IMAC buffer (GE Healthcare). v) β-DDM]. Mzerewo unatsukidwa ndi ma CV asanu a IMAC buffer, kenako ndi ma CV asanu a IMAC buffer okhala ndi 10 mM histidine. Core complex inachotsedwa pa chigawocho ndi ma buffer asanu a IMAC okhala ndi 100 mM histidine. Gawo lomwe lili ndi RC-LH114-W complex limayikidwa mpaka ~10 ml mu thanki yosunthidwa yokhala ndi fyuluta ya Amicon 100,000 MWCO (Merck, UK), kuchepetsedwa nthawi 20 ndi buffer yomangira, kenako kuwonjezeredwa ku 25 ml Mu DEAE Sepharose column, ma CV anayi omangiriridwa ku buffer amagwiritsidwa ntchito pasadakhale. Tsukani mzati ndi ma buffer anayi omangira a CV, kenako chotsani complex pa ma CV asanu ndi atatu pa mzere wolunjika wa 0 mpaka 100 mM NaCl (mu chomangira chomangira), ndi ma CV anayi otsala okhala ndi 100 mM chomangira chomangira. Ma complex otsala omwe adachotsedwa pa sodium chloride pamodzi ndi chiŵerengero cha A880/A280 choposa 2.4 ndi chiŵerengero cha A880/A805 choposa 4.6 fractions adayikidwa mpaka ~2 ml mu fyuluta ya Amicon 100,000 MWCO centrifugal, ndikudzazidwa ndi 1.5 CV IMAC pasadakhale Buffer equilibrated Superdex 200 16/600 size exclusion column, kenako chochotsedwa mu chomangira chomwecho pa 1.5 CV. Sonkhanitsani ma spectra oyamwa a magawo olekanitsa kukula ndi kuyika ma spectra oyamwa ndi ma A880/A280 ratios opitilira 2.1 ndi ma A880/A805 ratios opitilira 4.6 mpaka 100 A880, omwe amagwiritsidwa ntchito nthawi yomweyo pokonzekera gridi ya TEM yozizira kapena kusungidwa pa -80°C mpaka pakufunika.
Chotsukira madzi cha Leica EM GP chinagwiritsidwa ntchito pokonza ma gridi a TEM otentha kwambiri. Chotsukiracho chinasungunuka mu IMAC buffer kufika pa A880 ya 50, kenako 5μl inayikidwa pa QUANTIFOIL 1.2/1.3 copper-coated coated mesh yatsopano (Agar Scientific, UK). Ikani gridi pa 20°C ndi chinyezi cha 60% kwa masekondi 30, kenako iume kwa masekondi atatu, kenako ichotseni mu ethane yamadzimadzi pa -176°C.
Deta ya RC-LH114-W complex inalembedwa pa eBIC (Electronic Bioimaging Center) (British Diamond Light Source) ndi maikulosikopu ya Titan Krios, yomwe imagwira ntchito pa voltage yothamanga ya 300kV, yokhala ndi kukula kwapadera kwa 130,000× ndi mphamvu ya- Sankhani kusiyana kwa 20 eV. Gatan 968 GIF Quantum yokhala ndi K2 peak detector inagwiritsidwa ntchito kujambula zithunzi mu njira yowerengera kuti isonkhanitse deta. Kukula kwa pixel yolinganizidwa ndi 1.048Å, ndipo mlingo wa mlingo ndi 3.83 e-Å-2s-1. Ndinasonkhanitsa filimuyo mu masekondi 11 ndikuigawa m'magawo 40. Gwiritsani ntchito dera lophimbidwa ndi kaboni kuti muyang'anenso maikulosikopu, kenako sonkhanitsani makanema atatu pa dzenje lililonse. Zonse, makanema 3130 anasonkhanitsidwa, ndi ma values ​​​​osayang'ana pakati pa -1 ndi -3μm.
Deta ya RC-LH116 complex inasonkhanitsidwa pogwiritsa ntchito maikulosikopu yomweyo ku Asterbury Biostructure Laboratory (University of Leeds, UK). Detayo inasonkhanitsidwa mu njira yowerengera ndi kukula kwa 130 k, ndipo kukula kwa pixel kunalinganizidwa kufika pa 1.065 Å ndi mlingo wa 4.6 e-Å-2s-1. Filimuyo inajambulidwa mu masekondi 12 ndipo inagawidwa m'magawo 48. Ponseponse, mafilimu 3359 anasonkhanitsidwa, ndi ma values ​​​​osayang'ana pakati pa -1 ndi -3μm.
Kukonza deta konse kumachitika mu Relion 3.0 pipeline (42). Gwiritsani ntchito Motioncorr 2 (43) kuti mukonze kayendedwe ka beam poyesa kuchuluka kwa dose, kenako gwiritsani ntchito CTFFIND 4.1 (44) kuti mudziwe CTF (contrast transfer function). Ma photomicrographs wamba pambuyo pa magawo oyamba awa akuwonetsedwa mu Chithunzi 2. S16. Chifaniziro chosankha chokha chimapangidwa mwa kusankha pamanja ma pixel pafupifupi 250 a tinthu 1000 mu chimango cha ma pixel 250 komanso palibe gulu la ma 2D, motero kukana magulu omwe amakumana ndi kuipitsidwa kwa zitsanzo kapena omwe alibe mawonekedwe owoneka. Kenako, kusankha kokha kunachitika pa ma microphotographs onse, ndipo RC-LH114-W inali tinthu 849,359, ndipo RC-LH116 complex inali tinthu 476,547. Tinthu tonse tosankhidwa tadutsa m'magulu awiri osatchulidwa a 2D, ndipo pambuyo pa kuthamanga kulikonse, tinthu tomwe timakumana ndi dera la kaboni, kuipitsidwa kwa zitsanzo, palibe zinthu zoonekeratu kapena tinthu tomwe timalumikizana kwambiri timakanidwa, zomwe zimapangitsa kuti tinthu 772,033 (90.9%) ndi 359,678 (75.5%) tigwiritsidwe ntchito pogawa RC-LH114-W ndi RC-LH116 mu 3D motsatana. Chitsanzo choyamba cha 3D chinapangidwa pogwiritsa ntchito njira yotsika ya stochastic gradient. Pogwiritsa ntchito chitsanzo choyamba ngati chofotokozera, tinthu tosankhidwa timagawidwa m'magulu anayi mu 3D. Pogwiritsa ntchito chitsanzo chomwe chili m'gululi ngati chofotokozera, chitani kuyeretsa kwa 3D pa tinthu tating'onoting'ono tomwe tili m'gulu lalikulu kwambiri, kenako gwiritsani ntchito fyuluta yoyambira ya 15Å low-pass kuti muphimbe malo osungunulira, onjezani ma pixel 6 a m'mphepete zofewa, ndikukonza ma pixel kuti mukonze ntchito yosinthira ya Gatan K2 peak Modulation ya chowunikira chapamwamba. Pa deta ya RC-LH114-W, chitsanzo choyambachi chinasinthidwa pochotsa kuchuluka kwamphamvu m'mphepete mwa chigoba (chosalumikizidwa ku kuchuluka kwa core complex mu UCSF Chimera). Ma model omwe amachokera (ma resolution a RC-LH114-W ndi RC-LH116 ndi 3.91 ndi 4.16 Å, motsatana) amagwiritsidwa ntchito ngati chisonyezero cha gawo lachiwiri la gulu la 3D. Tinthu tomwe timagwiritsidwa ntchito timagawidwa m'gulu loyamba la 3D ndipo tilibe mgwirizano wamphamvu ndi dera. Kuphatikizika kapena kusowa kwa mawonekedwe omveka bwino a kapangidwe kake. Pambuyo pa gawo lachiwiri la gulu la 3D, gulu lomwe lili ndi resolution yapamwamba kwambiri linasankhidwa [Pa RC-LH114-W, gulu limodzi ndi tinthu ta 377,703 (44.5%), pa RC-LH116, pali magulu awiri, onse tinthu ta 260,752 (54.7%), komwe amakhala ofanana pokhapokha akalumikizidwa pambuyo pa kuzungulira koyamba ndi kusiyana pang'ono]. Tinthu tomwe tasankhidwa timachotsedwanso m'bokosi la ma pixel 400 ndikukonzedwanso ndi 3D refine. Chigoba chosungunula chimapangidwa pogwiritsa ntchito fyuluta yoyambira ya 15Å low-pass, kukulitsa mapu a ma pixel 3 ndi chigoba chofewa cha ma pixel 3. Pogwiritsa ntchito CTF refinement pa tinthu tating'onoting'ono, kukonza kayendedwe ka tinthu tating'onoting'ono ndi kuzungulira kwachiwiri kwa CTF refinement pa tinthu tating'onoting'ono, 3D refinement, solvent masking ndi post-processing zimachitika pambuyo pa sitepe iliyonse kuti ziwongolere kapangidwe kake. Pogwiritsa ntchito FSC (Fourier Shell Correlation Coefficient) cut-off value ya 0.143, ma resolution a ma models omaliza a RC-LH114-W ndi RC-LH116 ndi 2.65 ndi 2.80Å, motsatana. FSC curve ya chitsanzo chomaliza ikuwonetsedwa pa Chithunzi 2. S17.
Ma sequence onse a mapuloteni amatsitsidwa kuchokera ku 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); Puloteni-W (PufW; UniProt ID: Q6N1K3). SWISS-MODEL (45) idagwiritsidwa ntchito popanga chitsanzo cha homology cha RC, chomwe chili ndi ma sequence a mapuloteni a RC-L, RC-M ndi RC-H ndipo kapangidwe ka kristalo ka Rba. sphaeroides RC idagwiritsidwa ntchito ngati template (PDB ID: 5LSE) (46). Gwiritsani ntchito chida cha "fit map" mu UCSF Chimera kuti mugwirizane ndi chitsanzo chopangidwacho ndi mapu (47), sinthani kapangidwe ka mapuloteni, ndipo cofactor [4×BChl a (dzina la residue la monomer library = BCL), 2×BPh a (BPH), mtundu umodzi kapena iwiri ya UQ10 (U10), umodzi wosakhala heme iron (Fe) ndi umodzi wa 3,4-dihydrohexacarbonylcholine (QAK)] gwiritsani ntchito Coot (48) kuwonjezera. Popeza QAK siipezeka mu laibulale ya monomer, idasinthidwa pogwiritsa ntchito chida cha eLBOW mu PHENIX (49).
Kenako, gawo la LH1 linapangidwa. Poyamba, chida chomangira chokha mu PHENIX (49) chinagwiritsidwa ntchito kupanga gawo la gawo la LH1 pogwiritsa ntchito mapu ndi ma protein a LH1-α ndi LH1-β ngati cholowera. Sankhani gawo lathunthu la LH1, lichotseni ndikuliyika mu Coot, onjezerani pamanja gawo lomwe likusowa, ndikuyeretsa kapangidwe kake konse musanawonjezere ma BCls awiri a (BCL) ndi spirilloxanthin (CRT) [malinga ndi kuchuluka kwa Rps komwe kuli koyenera kwa gulu la LH1 ndi kuchuluka kwa carotenoid komwe kumadziwika. Mitundu (17)]. Koperani gawo lonse la LH1, ndikugwiritsa ntchito UCSF Chimera "Docking Map Tool" kuti muyike pamalo oyandikana ndi omwe si a chitsanzo a kuchuluka kwa LH1, kenako muyiyeretse mu Coot; bwerezani njirayi mpaka magawo onse a LH1 atapangidwa. Pa kapangidwe ka RC-LH114-W, pochotsa kuchuluka kwa mapuloteni osagawika mu Coot, puloteniyo imagawidwa kuchokera ku zigawo zotsala zomwe sizili mapuloteni mu USCF Chimera map ndipo chida cha Autobuild chimagwiritsidwa ntchito kukhazikitsa chitsanzo choyambirira, ndi ma subunits otsala (protein-W) Modeling. Mu PHENIX (49). Onjezani ma sequences aliwonse omwe akusowa ku chitsanzo chomwe chatuluka mu Coot (48), kenako sinthani pamanja subunit yonse. Kuchuluka kwa mafuta osagawika kotsala kumagwirizana ndi kuphatikiza kwa lipids (PDB monomer library ID ya CDL = CDL, POPC = 6PL ndi POPG = PGT), β-DDM detergent (LMT) ndi mamolekyu a UQ10 (U10). Gwiritsani ntchito PHENIX optimization (49) ndi manual optimization mu Coot (48) kuti mukonze chitsanzo chonse choyambirira mpaka ziwerengero za chitsanzo ndi mawonekedwe a mawonekedwe a fit zisapitirire kukonzedwa. Pomaliza, gwiritsani ntchito LocScale (50) kuti muwongolere mapu am'deralo, kenako chitani njira zina zingapo zowonetsera kuchuluka kwa mafuta osagawika komanso kukonza kwa automatic ndi manual.
Ma peptide, ma cofactor ndi ma lipid ndi ma quinone ena omwe ali mkati mwa kuchuluka kwawo akuwonetsedwa mu Zithunzi 1 ndi 2. S18 mpaka S23. Zambiri za ziwerengero za chitsanzo chomaliza zikuwonetsedwa mu Table S1.
Pokhapokha ngati patchulidwa mwanjira ina, ma spectra a UV/Vis/NIR absorption adasonkhanitsidwa pa Cary60 spectrophotometer (Agilent, USA) pa 1 nm intervals kuyambira 250 nm mpaka 1000 nm ndi nthawi yolumikizira ya 0.1s.
Sakanizani chitsanzocho mu quartz cuvette yokhala ndi njira ya 2 mm kupita ku A880 ya 1, ndikusonkhanitsa ma absorption spectrum pakati pa 400 ndi 1000 nm. Ma circular dichroic spectra adasonkhanitsidwa pa Jasco 810 spectropolarimeter (Jasco, Japan) pa intervals ya 1 nm pakati pa 400 nm ndi 950 nm pa scan rate ya 20 nm min-1.
Kuchuluka kwa molar extinction coefficient kumatsimikiziridwa mwa kuchepetsa core complex kufika pa A880 ya pafupifupi 50. Sakanizani voliyumu ya 10μl mu 990μl binding buffer kapena methanol, ndikusonkhanitsa spectrum yoyamwa nthawi yomweyo kuti muchepetse kuwonongeka kwa BChl. Kuchuluka kwa BChl mu chitsanzo chilichonse cha methanol kunawerengedwa ndi extinction coefficient pa 771 nm ya 54.8 mM-1 cm-1, ndipo extinction coefficient idatsimikiziridwa (51). Gawani kuchuluka kwa BChl komwe kunayesedwa ndi 32 (RC-LH114-W) kapena 36 (RC-LH116) kuti mudziwe kuchuluka kwa core complex, komwe kumagwiritsidwa ntchito kudziwa ma absorption spectrum a chitsanzo chomwecho chomwe chinasonkhanitsidwa mu buffer Extinction coefficient. parallel. Miyeso itatu yobwerezabwereza idatengedwa pa chitsanzo chilichonse, ndipo avereji ya absorbance ya BChl Qy maximum idagwiritsidwa ntchito powerengera. Chiŵerengero cha kutha kwa RC-LH114-W choyezedwa pa 878 nm ndi 3280±140 mM-1 cm-1, pomwe chiŵerengero cha kutha kwa RC-LH116 choyezedwa pa 880 nm ndi 3800±30 mM-1 cm-1.
UQ10 inayesedwa malinga ndi njira yomwe ili mu (52). Mwachidule, HPLC ya reverse phase (RP-HPLC) idachitika pogwiritsa ntchito njira ya Agilent 1200 HPLC. Sungunulani pafupifupi 0.02 nmol ya RC-LH116 kapena RC-LH114-W mu 50μl ya 50:50 methanol:chloroform yokhala ndi 0.02% (w/v) ferric chloride, ndikulowetsa Beckman Coulter Ultrasphere ODS 4.6 mm Sungunulani mu 1 ml-1 min-1 pa 40°C mu HPLC solvent (80:20 methanol:2-propanol) pa ×25 cm column. Chitani isocratic elution mu HPLC solvent kuti muwone kuyamwa pa 275 nm (UQ10), 450 nm (carotenoids) ndi 780 nm (BChl) kwa ola limodzi. Chiwongola dzanja cha 275 nm chromatogram pa mphindi 25.5 chinaphatikizidwa, chomwe sichinali ndi mankhwala ena aliwonse omwe angapezeke. Malo ophatikizidwa amagwiritsidwa ntchito kuwerengera kuchuluka kwa molar kwa UQ10 komwe kunachotsedwa potengera kukhazikika kwa calibration komwe kunawerengedwa kuchokera ku jakisoni wa miyezo yoyera kuyambira 0 mpaka 5.8 nmol (Chithunzi S14). Chitsanzo chilichonse chinasanthulidwa m'magawo atatu, ndipo cholakwika chomwe chinanenedwacho chikugwirizana ndi SD ya avareji.
Yankho lokhala ndi RC-LH1 complex yokhala ndi Qy absorption yayikulu ya 0.1 linakonzedwa ndi 30 μM reduced horse heart cytochrome c2 (Merck, UK) ndi 0 mpaka 50 μMUQ2 (Merck, UK). Zitsanzo zitatu za 1-ml zinakonzedwa pa UQ2 concentration iliyonse ndipo zinayikidwa usiku wonse mumdima pa 4°C kuti zitsimikizire kuti zikugwirizana bwino ndi mdima musanayese. Yankholo linayikidwa mu OLIS RSM1000 modular spectrophotometer yokhala ndi 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm middle ndi 0.6 mm outlet slits. Fyuluta yodutsa ya 600 nm yayitali imayikidwa pakhomo la phototube ya chitsanzo ndi chubu chowunikira photomultiplier kuti muchotse kuwala kosangalatsa. Kuyamwa kunayang'aniridwa pa 550 nm ndi nthawi yolumikizira ya 0.15 masekondi. Kuwala koyambitsa kumatulutsa kuwala kuchokera ku LED ya 880 nm M880F2 (Light Emitting Diode) (Thorlabs Ltd., UK) kudzera mu chingwe cha fiber optic pa 90% mphamvu kudzera mu chowongolera cha DC2200 (Thorlabs Ltd., UK) ndipo kumatulutsidwa ku gwero la kuwala pa ngodya ya 90° ya. Mzere woyezera umatsutsana ndi galasi kuti ubwezeretse kuwala kulikonse komwe sikunalowereredwe ndi chitsanzo poyamba. Yang'anirani kuyamwa kwa masekondi 10 musanawone kuwala kwa masekondi 50. Kenako kuyamwa kunayang'aniridwanso kwa masekondi 60 mumdima kuti muwone momwe quinolol imachepetsera cytochrome c23 + mwangozi (onani Chithunzi S8 kuti mudziwe zambiri).
Detayo idakonzedwa mwa kuyika liwiro loyambira la mzere mkati mwa masekondi 0.5 mpaka 10 (kutengera kuchuluka kwa UQ2) ndikuwerengera kuchuluka kwa zitsanzo zonse zitatu pa kuchuluka kulikonse kwa UQ2. Kuchuluka kwa RC-LH1 komwe kudawerengedwa ndi coefficient yofananira kudagwiritsidwa ntchito kusintha liwiro kukhala catalytic efficiency, yojambulidwa mu Origin Pro 2019 (OriginLab, USA), ndikuyikidwa ku chitsanzo cha Michaelis-Menten kuti adziwe zomwe zikuwonekera Km ndi Kcat values.
Pakuyeza kwa kuyamwa kwa transient, chitsanzo cha RC-LH1 chinachepetsedwa kufika pa ~2μM mu IMAC buffer yokhala ndi 50 mM sodium ascorbate (Merck, USA) ndi 0.4 mM Terbutin (Merck, USA). Ascorbic acid imagwiritsidwa ntchito ngati wopereka ma electron odzipereka, ndipo tert-butaclofen imagwiritsidwa ntchito ngati choletsa QB kuti zitsimikizire kuti wopereka wamkulu wa RC akucheperachepera (ndiko kuti, osati photooxidized) panthawi yonse yoyezera. Pafupifupi 3 ml ya chitsanzo imawonjezedwa ku selo yozungulira yokhazikika (pafupifupi 0.1 m m'mimba mwake, 350 RPM) yokhala ndi kutalika kwa njira ya 2 mm optical kuti zitsimikizire kuti chitsanzo chomwe chili mu njira ya laser chili ndi nthawi yokwanira yosinthira mdima pakati pa ma pulse oyambitsa. Gwiritsani ntchito ma pulse a laser a ~100-fs kuti muwonjezere dongosolo la laser la Ti: Sapphire (Spectra Physics, USA) kuti musangalatse chitsanzocho pa 880 nm pamlingo wobwerezabwereza wa 1 kHz (20 nJ ya NIR kapena 100 nJ ya Vis). Musanasonkhanitse deta, ikani chitsanzocho ku kuwala kochititsa chidwi kwa mphindi pafupifupi 30. Kuwonekera kudzapangitsa kuti QA isagwire ntchito (mwina kuchepetsa QA kamodzi kapena kawiri). Koma chonde dziwani kuti njirayi ndi yosinthika chifukwa pambuyo pa nthawi yayitali yosintha mdima, RC idzabwerera pang'onopang'ono ku ntchito ya QA. Helios spectrometer (Ultrafast Systems, USA) idagwiritsidwa ntchito poyesa ma spectra osinthasintha ndi nthawi yochedwa ya -10 mpaka 7000 ps​​. Gwiritsani ntchito pulogalamu ya Surface Xplorer (Ultrafast Systems, USA) kuti muchotse ma data, kenako muphatikize ndikuyiyika muyeso. Gwiritsani ntchito pulogalamu ya CarpetView (Light Conversion Ltd., Lithuania) kuti mugwiritse ntchito deta yophatikizana kuti mupeze ma spectra osiyana okhudzana ndi kuwola, kapena gwiritsani ntchito ntchito yomwe imagwiritsa ntchito ma exponents angapo ndi yankho la chida kuti ligwirizane ndi kusintha kwa ma spectral a single-wavelength ku Origin (OriginLab, USA).
Monga tafotokozera pamwambapa (53), filimu yopangira photosynthesis yokhala ndi LH1 complex yopanda RC ndi peripheral LH2 antenna inakonzedwa. Nembanembayo inachepetsedwa mu 20 mM tris (pH 8.0) kenako inayikidwa mu quartz cuvette yokhala ndi njira yowunikira ya 2 mm. Kugunda kwa laser kwa 30nJ kunagwiritsidwa ntchito kusangalatsa chitsanzocho pa 540 nm ndi nthawi yochedwetsa ya -10 mpaka 7000 ps. Chitani zomwe zasungidwa monga momwe zafotokozedwera chitsanzo cha Rps. pal.
Nembanembayo inalowetsedwa ndi centrifugation pa 150,000 RCF kwa maola awiri pa 4°C, kenako kuyamwa kwake pa 880 nm kunabwezeretsedwanso mu 20 mM tris-HCl (pH 8.0) ndi 200 mM NaCl. Sungunulani nembanembayo posakaniza pang'onopang'ono 2% (w/v) β-DDM kwa ola limodzi mumdima pa 4°C. Chitsanzocho chinachepetsedwa mu 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) mpaka kuchuluka kwa mapuloteni a 2.5 mg ml-1 (Bio-Rad analysis). Kukonza kwina kunachitika kuchokera ku njira yomwe idasindikizidwa kale (54), kuyambira ndi kuchepetsedwa kwa 50 μg protein kukhala 50 μl TEAB yokhala ndi 1% (w/v) sodium laurate (Merck, UK). Pambuyo pa sonication kwa masekondi 60, idachepetsedwa ndi 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) pa 37°C kwa mphindi 30. Pa S-alkylation, ikani chitsanzocho ndi 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) ndikuchiwonjezera kuchokera ku yankho la isopropanol stock la 200 mM kwa mphindi 10 kutentha kwa chipinda. Kugaya kwa proteolytic kunachitika powonjezera 2 μg trypsin/endoproteinase Lys-C mixture (Promega UK) ndikuyikidwa pa 37°C kwa maola 3. Laurate surfactant idachotsedwa powonjezera 50 μl ethyl acetate ndi 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) ndikutulutsa vortex kwa masekondi 60. Kulekanitsa magawo kunalimbikitsidwa ndi centrifugation pa 15,700 RCF kwa mphindi 5. Malinga ndi njira ya wopanga, column ya C18 spin (Thermo Fisher Scientific, UK) idagwiritsidwa ntchito kupopera mosamala ndikuchotsa mchere mu gawo lotsika lomwe lili ndi peptide. Pambuyo pouma pogwiritsa ntchito vacuum centrifugation, chitsanzocho chinasungunuka mu 0.5% TFA ndi 3% acetonitrile, ndipo 500 ng idasanthulidwa ndi nanoflow RP chromatography yolumikizidwa ndi mass spectrometry pogwiritsa ntchito magawo a system omwe afotokozedwa kale.
Gwiritsani ntchito MaxQuant v.1.5.3.30 (56) kuti mudziwe mapuloteni ndi kuchuluka kwake kuti mufufuze Rps. palustris proteome database (www.uniprot.org/proteomes/UP000001426). Deta ya mass spectrometry proteomics yasungidwa mu ProteomeXchange Alliance kudzera mu PRIDE partner repository (http://proteomecentral.proteomexchange.org) pansi pa deta ya PXD020402.
Kuti mufufuze ndi RPLC pamodzi ndi electrospray ionization mass spectrometry, RC-LH1 complex idapangidwa kuchokera ku wild-type Rps. Pogwiritsa ntchito njira yomwe idasindikizidwa kale (16), kuchuluka kwa mapuloteni omwe amapangidwa m'maselo a palustris kunali 2 mg ml-1 mu 20 mM Hepes (pH 7.8), 100 mM NaCl ndi 0.03% (w/v) β- (Bio-Rad analysis) ) DDM. Malinga ndi njira ya wopanga, gwiritsani ntchito 2D purification kit (GE Healthcare, USA) kuti mutulutse 10 μg protein pogwiritsa ntchito njira ya precipitation, ndikusungunula precipitate mu 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile ndi 20% (v/v) madzi. Ma microliters asanu adawunikidwa ndi RPLC (Dionex RSLC) pamodzi ndi mass spectrometry (Maxis UHR-TOF, Bruker). Gwiritsani ntchito MabPac 1.2×100 mm column (Thermo Fisher Scientific, UK) kuti mulekanitse kutentha pa 60°C ndi 100μlmin -1, yokhala ndi gradient ya 85% (v / v) solvent A [0.1% (v / v) FA ndi 0.02% (V/v) TFA aqueous solution] ku 85%(v/v) solvent B [0.1%(v/v) FA ndi 0.02%(v/v) mu 90%(v/v) acetonitrile TFA] Pogwiritsa ntchito standard electrospray ionization source ndi default parameters kwa mphindi zoposa 60, mass spectrometer imapeza 100 mpaka 2750 m/z (mass-to-charge ratio). Mothandizidwa ndi ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), jambulani mass spectrum ku ma subunits a complex.
Maselo anakula kwa maola 72 pansi pa 100 ml NF-low (10μMm-2 s-1), yapakati (30μMm-2 s-1) kapena yapamwamba (300μMm-2 s-1). M22 yapakati (M22 yapakati yomwe ammonium sulfate imasiyidwa ndipo sodium succinate imalowetsedwa ndi sodium acetate) mu botolo la 100 ml screw-top (23). Mu magawo asanu a 30-sekondi, mikanda yagalasi ya 0.1 micron inakulungidwa pa chiŵerengero cha voliyumu cha 1:1 kuti ipange maselo ndikuzizira pa ayezi kwa mphindi 5. Zinthu zosasungunuka, maselo osasweka ndi mikanda yagalasi zinachotsedwa ndi centrifugation pa 16,000 RCF kwa mphindi 10 mu benchtop microcentrifuge. Nembanembayo inalekanitsidwa mu rotor ya Ti 70.1 yokhala ndi 100,000 RCF mu 20 mM tris-HCl (pH 8.0) yokhala ndi 40/15% (w/w) sucrose gradient kwa maola 10.
Monga tafotokozera m'ntchito yathu yapitayi, kuzindikira chitetezo cha mthupi cha chizindikiro cha His pa PufW (16). Mwachidule, puloteni yoyeretsedwa (11.8 nM) kapena nembanemba yokhala ndi kuchuluka komweko kwa RC (yomwe imatsimikiziridwa ndi oxidation kuchotsa kusiyana kochepetsedwa ndikufananiza katundu pa gel yopaka) mu 2x SDS loading buffer (Merck, UK) idachepetsedwa kawiri. Mapuloteniwo adalekanitsidwa pa replica 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). Gel idapakidwa utoto ndi Coomassie Brilliant Blue (Bio-Rad, UK) kuti ikweze ndikuwona RC-L subunit. Puloteni pa gel yachiwiri idasamutsidwira ku nembanemba ya methanol-activated polyvinylidene fluoride (PVDF) (Thermo Fisher Scientific, UK) kuti igwiritsidwe ntchito poyesa chitetezo chamthupi. Nembanemba ya PVDF inatsekedwa mu 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 ndi 5% (w / v) ufa wa mkaka wochotsedwa mafuta, kenako inayikidwa ndi anti-His primary antibody (mu Dilute the antibody buffer [50 mM tris-HCl (pH 7.6), 150 mM NaCl ndi 0.05% (v/v) Tween-20] mu 1:1000 A190-114A, Bethyl Laboratories, USA) kwa maola anayi. Pambuyo potsuka katatu kwa mphindi 5 mu antibody buffer, nembanembayo inaphatikizidwa ndi horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (yochepetsedwa 1:10,000 mu antibody buffer). Ikani mu incubate kuti ipezeke (mphindi 5 mutatsuka katatu mu antibody buffer) pogwiritsa ntchito WESTAR ETA C 2.0 chemiluminescence substrate (Cyanagen, Italy) ndi Amersham Imager 600 (GE Healthcare, UK).
Mwa kujambula kufalikira kwa mphamvu ya gel iliyonse yopaka utoto kapena njira yoyesera chitetezo cha mthupi, kuphatikiza malo omwe ali pansi pa nsonga ndikuwerengera chiŵerengero cha mphamvu ya RC-L (gel yopaka utoto) ndi Protein-W (immunoassay), mu ImageJ (57) Chitani chithunzicho. Ma ratios awa adasinthidwa kukhala ma molar ratios poganiza kuti chiŵerengero cha RC-L ndi protein-W mu chitsanzo choyera cha RC-LH114-W chinali 1:1 ndikukonzanso deta yonse moyenerera.
Kuti mudziwe zambiri zowonjezera pa nkhaniyi, chonde onani http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
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David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe, Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Kapangidwe kake ka light trap 1 complex kokhala ndi mawonekedwe apamwamba kwambiri komwe kali mkati mwa reaction center kamapereka chidziwitso chatsopano cha mphamvu ya 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
Kapangidwe kake ka light trap 1 complex kokhala ndi mawonekedwe apamwamba kwambiri komwe kali mkati mwa reaction center kamapereka chidziwitso chatsopano cha mphamvu ya quinone.
©2021 American Association for the Advancement of Science. maumwini onse ndi otetezedwa. AAAS ndi mnzake wa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef ndi COUNTER. ScienceAdvances ISSN 2375-2548.