Tam sim no†Chaw nyob tam sim no: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Lub chaw tshuaj tiv thaiv teeb pom kev zoo 1 (RC-LH1) yog lub hauv paus tseem ceeb ntawm cov kab mob phototrophic ntshav. Peb tau qhia ob lub qauv cryo-electron microscopy ntawm RC-LH1 complex los ntawm Rhodopseudomonas palustris. Lub qauv daws teeb meem 2.65-Å ntawm RC-LH114-W complex muaj 14 subunit LH1 loops nyob ib puag ncig RC, uas raug cuam tshuam los ntawm protein W, thaum lub complex tsis muaj protein-W yog RC sib xyaw ua ke tag nrho nyob ib puag ncig los ntawm RC. Kaw 16 subunit LH1 loop. Kev sib piv ntawm cov qauv no muab kev nkag siab rau hauv dynamics ntawm quinone hauv RC-LH1 complex, suav nrog cov kev hloov pauv conformational tsis tau txiav txim siab thaum khi quinone ntawm RC QB qhov chaw, nrog rau qhov chaw ntawm cov chaw khi quinone pabcuam, uas pab dhau lawv mus rau RC. Lub qauv tshwj xeeb ntawm W protein tiv thaiv kev kaw ntawm LH1 loop, yog li tsim ib txoj hauv kev rau kev ua kom nrawm dua quinone / quinolone pauv.
Lub zog uas muab los ntawm photosynthesis tuaj yeem txhawb nqa yuav luag txhua yam tsiaj txhu hauv ntiaj teb, thiab nws muaj peev xwm zoo rau lub hnub ci biotechnology. Thaum txhawb nqa kev tsim photosynthesis thoob ntiaj teb, cov kab mob phototrophic xim av kuj qhia txog ntau hom zog thiab kev muaj peev xwm ua kom lub cev zom zaub mov zoo. Lawv tuaj yeem zam kev tsim photosynthesis thiab loj hlob ua cov kab mob heterotrophic hauv qhov tsaus ntuj, tuaj yeem kho nitrogen thiab carbon dioxide, tsim hydrogen, thiab rhuav tshem cov tshuaj aromatic (1-3). Yuav kom muab lub zog rau cov txheej txheem no, lub teeb yuav tsum tau hloov pauv sai thiab zoo mus rau hauv lub zog tshuaj lom neeg. Cov txheej txheem no pib thaum lub teeb-ntxiab antenna complex nqus lub teeb thiab hloov lub zog ntes mus rau qhov chaw tshuaj tiv thaiv (RC), yog li pib sib cais them (4 - 7). Lub hauv paus ntawm photosynthesis hauv cov kab mob phototrophic xim av yog tsim los ntawm hom 2 RC, nyob ib puag ncig los ntawm lub teeb-harvesting complex 1 (LH1), tsim cov RC-LH1 core complex. LH1 yog tsim los ntawm ib qho array ntawm curved αβ heterodimers, txhua tus uas khi ob lub kab mob chlorophyll (BChl) a molecules thiab ib lossis ob lub carotenoids (8-12). Lub kav hlau txais xov LH1 yooj yim tshaj plaws muaj 16 lossis 17 αβ heterodimers uas nyob ib puag ncig RC (9-13) hauv lub voj kaw, tab sis hauv lwm cov tub ntxhais tseem ceeb, transmembrane peptides cuam tshuam qhov kev sib txuas ntawm LH1 ib puag ncig, Yog li txhawb nqa quinol / quinone diffusion ntawm RC thiab cytochrome bc1 complex (11, 13-15). Cov nroj tsuag phototrophic xim av Rhodopseudomonas (Rps.) yog ib qho qauv kab mob uas tuaj yeem nkag siab txog lub zog thiab kev hloov pauv hluav taws xob uas txhawb nqa photosynthesis. Thawj cov qauv siv lead ua ntawm Rps. Tus qauv ntawm palustris RC-LH1 complex yog RC, nyob ib puag ncig los ntawm 15 heterodimeric LH1 loops, uas raug cuam tshuam los ntawm cov protein tsis paub hu ua "Protein W" (14). Protein-W tom qab ntawd tau txheeb xyuas tias yog RPA4402, uas yog ib qho protein 10.5kDa tsis tau cim nrog peb qhov kwv yees transmembrane helices (TMH) (16). Peb xav kom hloov lub npe ntawm rpa4402 gene encoding protein W mus rau pufW kom sib xws nrog lub npe siv rau cov genes encoding RC-L, M (pufL, pufM) thiab LH1α, β (pufA, pufB) subunits. Qhov nthuav yog, protein-W tsuas yog muaj nyob rau hauv kwv yees li 10% ntawm RC-LH1, qhia Rps. palustris tsim ob lub complexes RC-LH1 sib txawv. Ntawm no, peb tshaj tawm cov qauv cryo-EM (cryo-EM) siab daws teeb meem ntawm ob lub complexes tseem ceeb, ib qho nrog protein W thiab 14 αβ heterodimers, lwm qhov tsis muaj protein W thiab kaw 16 Heterodimer LH1 voj voog. Peb cov qauv sawv cev rau kev hloov pauv kauj ruam hauv kev nkag siab ntawm RC-LH1 complex ntawm Rps. palustris, vim peb tau tshuaj xyuas cov pej xeem sib xws ntawm txhua qhov sib txawv thiab muaj kev daws teeb meem txaus los qhia meej txog txhua peptide thiab cov xim khi thiab cov lipids thiab quinones cuam tshuam. Qhov kev sib piv ntawm cov qauv no qhia tau hais tias peb cov TMH proteins-W uas tsis tau pom muaj nyob rau hauv lwm yam RC-LH1 complex txog tam sim no tsim cov quinone channel los ua kom nrawm dua qhov kev sib pauv quinone/quinolone. Muaj ntau qhov chaw khaws cia lipid thiab quinone binding sites tau txheeb xyuas, thiab peb tau qhia txog kev hloov pauv tshiab tom qab kev sib xyaw ua ke ntawm quinone thiab RC, uas tej zaum yuav tsim nyog rau photosystem II (PSII) RC ntawm cov kab mob oxygenated phototrophic. Peb cov kev tshawb pom muab cov kev nkag siab tshiab rau hauv kinetics ntawm quinone/quinolone binding thiab pauv hauv RC-LH1 core complex ntawm cov kab mob ntshav phototrophic.
Yuav kom pab txhawb kev tshawb nrhiav kom ntxaws txog ob lub complexes pom hauv Rps. palustris, peb cais txhua RC-LH1 los ntawm cov txheej txheem biochemical. Cov protein W-deficient complex (tom qab no hu ua ΔpufW) tau raug ntxuav los ntawm cov kab mob uas tsis muaj pufW gene (16), thiab tsuas yog ib qho RC-LH1 complex tuaj yeem tsim tau. Cov protein W-muaj complex yog tsim los ntawm ib hom kab mob. Cov protein W ntawm cov kab mob no tau hloov kho nrog 10x His tag ntawm nws C-terminus, yog li cov protein W-muaj complex tuaj yeem ua ke nrog feem ntau cov protein W uas tsis muaj los ntawm kev immobilizing hlau. Cov complex tau raug cais tawm zoo (16) Affinity Chromatography (IMAC).
Raws li pom hauv Daim Duab 1, ob qho tib si muaj peb lub sub-unit RC (RC-L, RC-M thiab RC-H) nyob ib puag ncig los ntawm lub antenna LH1. Lub qauv 2.80-A ntawm lub complex uas tsis muaj protein-W qhia txog 16 αβ heterodimers, tsim ib lub voj voog LH1 kaw tag nrho nyob ib puag ncig RC, tom qab no hu ua RC-LH116 complex. Lub qauv 2.65Å ntawm lub complex uas muaj protein-W muaj 14-heterodimer LH1 uas raug cuam tshuam los ntawm protein-W, tom qab no hu ua RC-LH114-W.
(A thiab B) Sawv cev ntawm qhov chaw ntawm cov tshuaj. (C thiab D) Cov xim sib txuas uas qhia tawm hauv cov pas nrig. (E thiab F) Cov complexes pom los ntawm qhov chaw cytoplasmic muaj cov peptides thiab LH1 subunits sawv cev hauv cov duab kos, thiab raug suav clockwise los ntawm qhov sib txawv ntawm protein-W [sib xws nrog Rba tus lej. sphaeroides complex (13)]. Rau LH1-α, xim ntawm cov protein subunit yog daj; rau LH1-β, xim ntawm cov protein subunit yog xiav; rau protein-W, cov protein yog liab; rau RC-H, nws yog cyan; rau RC-L, nws yog txiv kab ntxwv; rau RC-M, magenta. Cofactors yog sawv cev los ntawm cov pas nrig, ntsuab sawv cev rau BChl thiab BPh a molecules, ntshav sawv cev rau carotenoids, thiab daj sawv cev rau UQ10 molecules. (G thiab H) Saib loj ntawm qhov sib txawv ntawm protein-W hauv thaj tsam sib npaug ntawm RC-LH114-W complex (G) thiab RC-LH116 complex (H). Cov cofactors tau tso tawm hauv daim ntawv ntawm qhov chaw sau, chelated quinone tau tso tawm hauv xiav. Qhov sib txawv ntawm protein-W tau qhia los ntawm kab xiav dashed hauv (G), thiab cov qhov me me uas quinone/quinolol diffuses ntawm lub nplhaib LH116 tau qhia los ntawm kab dub dashed hauv (H).
Daim Duab 1 (A thiab B) qhia txog RC uas muaj cov arrays qhib lossis kaw ntawm LH1αβ heterodimers, txhua tus uas khi ob BChl thiab ib carotenoid (Daim Duab 1, C thiab D). Cov kev tshawb fawb yav dhau los tau qhia tias Rps yog LH1 complex. Hauv txoj kev biosynthetic ntawm spirulina xanthin, cov tsiaj no muaj cov pej xeem sib xyaw ntawm carotenoids (17). Txawm li cas los xij, spiropyrroxanthin yog cov carotenoid tseem ceeb thiab nws qhov ceev yog qhov txaus siab. Yog li ntawd, peb tau xaiv los ua qauv spiroxanthin ntawm txhua qhov chaw khi LH1. Cov alpha thiab beta polypeptides yog cov TMHs ib leeg nrog cov cheeb tsam sab nraud luv luv (Daim Duab 1, A, B, E, thiab F). Txawm hais tias qhov ceev ntawm 17 residues ntawm C-terminus tsis tau pom, alpha polypeptide tau raug txiav los ntawm Met1 mus rau Ala46 hauv ob qho tib si complexes. β polypeptide tau txo qis los ntawm Gly4 mus rau Tyr52 hauv RC-LH116, thiab los ntawm Ser5 mus rau Tyr52 hauv RC-LH114-W. Tsis muaj qhov ceev ntawm 3 lossis 4 N-terminal lossis 13 C-terminal residues tau pom (Daim Duab S1). Kev tshuaj xyuas pawg spectrometry ntawm cov sib xyaw RC-LH1 complex uas tau npaj los ntawm cov kab mob qus qhia tau hais tias thaj chaw uas ploj lawm yog qhov tshwm sim ntawm kev sib cais ntawm cov peptides no (Daim Duab S1 thiab S2). N-terminal formylation ntawm α-Met1 kuj tau pom (f). Kev tshuaj xyuas qhia tau hais tias α-peptide muaj cov residues fMet1 rau Asp42/Ala46/Ala47/Ala50, thiab β-peptide muaj cov residues Ser2 rau Ala53, uas yog qhov zoo sib xws nrog daim ntawv qhia qhov ceev EM qis-kub.
Kev sib koom tes ntawm α-His29 thiab β-His36 ua rau BChls ntsej muag rau ntsej muag; txhua αβ heterodimer sib sau ua ke nrog nws cov neeg zej zog los tsim ib lub voj voog qhib (RC-LH114-W) lossis lub voj voog kaw (RC-LH116) nyob ib puag ncig RC Lub exciton txuas nrog pigment array (Daim Duab 1, C thiab D). Piv nrog rau 877 nm band ntawm RC-LH114-W, 880 nm absorption red shift ntawm RC-LH116 yog 3 nm (Daim Duab 2A). Txawm li cas los xij, lub voj voog dichroism spectrum yuav luag zoo ib yam (Daim Duab 2B), qhia tias txawm hais tias muaj qhov sib txawv meej ntawm qhib thiab kaw loops, qhov chaw ib puag ncig ntawm BChls zoo sib xws heev. Qhov absorption redshift tej zaum yuav yog qhov tshwm sim ntawm kev txo qis thermal txav thiab kev ruaj khov ntawm lub voj voog kaw (18, 19), qhov kev hloov pauv hauv pigment coupling tshwm sim los ntawm lub voj voog kaw (20, 21), lossis kev sib xyaw ua ke ntawm ob qho teebmeem no (11).
(A) Ultraviolet/pom tau/nyob ze-infrared absorption spectrum, cov peaks uas tau cim nrog lawv cov pigments sib xws thiab normalized rau BPh peak ntawm 775 nm. (B) Circular dichroism spectrum normalized rau BChl absorbance ntawm 805 nm. (C thiab D) Xaiv ΔA spectra los ntawm lub sijhawm-daws teeb meem absorption spectra ntawm RC-LH114-W complex (C) thiab RC-LH116 complex (D). Rau kev sib piv zoo dua, txhua spectra raug normalized rau ∆A ntawm −A ntawm 0.2 ps. (E) Tus nqi ntawm cytochrome c2 oxidation tom qab irradiation nyob rau hauv qhov muaj ntau yam concentration ntawm UQ2 (saib Daim Duab S8 rau cov ntaub ntawv raw). (F) Hauv cov hlwb loj hlob nyob rau hauv qis, nruab nrab lossis siab zog lub teeb (10, 30 lossis 300μMm-2 s-1, feem), cov protein W thiab RC-L subunits hauv lub purified complex thiab qhov sib cais membrane ratio. Txheeb xyuas qib protein los ntawm SDS-polyacrylamide gel electrophoresis thiab immunoassay (saib Daim Duab S9 rau cov ntaub ntawv raw). Txheeb xyuas qhov piv piv rau cov khoom siv RC-LH114-W uas tau ntxuav. Qhov piv stoichiometric ntawm RC-L rau protein-W ntawm cov khoom siv yog 1:1.
Cov BChls ntawm qhov chaw 1 hauv lub voj voog αβ14 deformed ntawm RC-LH114-W (Daim Duab 1, A, C, thiab E) nyob ze rau RC thawj tus neeg pub dawb (P) los ntawm 6.8Å dua li cov BChls sib npaug hauv RC-LH116 (Daim Duab 1, B, D, thiab F, thiab Daim Duab S3); txawm li cas los xij, lub sijhawm nqus dej ntawm ob lub complexes qhia tau tias rau RC-LH114-W thiab RC-LH116, lub sijhawm hloov pauv zog excitation los ntawm LH1 mus rau RC yog 40 ± 4 thiab 44 ± 3 ps (Daim Duab 2). , C thiab D, Daim Duab S4 thiab Rooj S2). Tsis muaj qhov sib txawv tseem ceeb hauv kev hloov pauv hluav taws xob hauv RC (Daim Duab S5 thiab cov ntawv ntxiv cuam tshuam). Peb xav tias qhov sib xws ze ntawm lub sijhawm hloov pauv zog ntawm LH1 thiab RC-P yog vim qhov deb, lub kaum sab xis thiab lub zog muaj peev xwm zoo sib xws ntawm feem ntau BChl hauv ob lub voj voog LH1. Zoo li kev tshawb nrhiav tus qauv zog LH1 kom mus txog qhov deb tsawg kawg nkaus tsis yog sai dua li kev hloov pauv zog ncaj qha los ntawm cov chaw tsis zoo mus rau RC. Lub voj voog qhib LH1 hauv RC-LH114-W kuj tseem yuav raug kev txav mus los tsis tseem ceeb hauv qab qhov kub qis rau kev tshuaj xyuas cov qauv, thiab muaj lub nplhaib αβ14 ntev dua ntawm qhov kub thiab txias ntawm chav tsev los ntawm qhov deb ntawm pigmentation ntawm βBChls ntawm qhov chaw ntawm RC 1.
Lub RC-LH116 complex muaj 32 BChls thiab 16 carotenoids, thiab nws qhov kev npaj tag nrho yog tib yam li qhov tau txais los ntawm Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 hom (PDB ID 7C9R) (12) thiab algae ntsuab (Blc.viridis) (PDB ID 6ET5) (10). Tom qab kev sib dhos, tsuas yog qhov sib txawv me me hauv qhov chaw ntawm αβ heterodimers tau pom, tshwj xeeb tshaj yog 1-5, 15, thiab 16 (Daim Duab S6). Qhov muaj protein-W muaj qhov cuam tshuam loj rau cov qauv ntawm LH1. Nws peb TMHs txuas nrog los ntawm cov voj luv luv, nrog N-terminal ntawm sab lumen ntawm lub complex thiab C-terminal ntawm sab cytoplasmic (Daim Duab 1A thiab 3, A txog D). Protein-W feem ntau yog hydrophobic (Daim Duab 3B), thiab TMH2 thiab TMH3 cuam tshuam nrog LH1αβ-14 los tsim ib qho chaw transmembrane (Daim Duab 3, B thiab E rau G). Lub interface feem ntau yog tsim los ntawm Phe, Leu thiab Val residues hauv thaj chaw transmembrane. Cov residues no tau sib sau ua ke nrog hydrophobic amino acids thiab αβ-14 pigments. Qee cov polar residues kuj pab txhawb rau kev sib cuam tshuam, suav nrog hydrogen bond ntawm W-Thr68 thiab β-Trp42 ntawm qhov chaw ntawm lub complex cavity (Daim Duab 3, F thiab G). Ntawm qhov chaw ntawm cytoplasm, Gln34 nyob ib sab ntawm keto pawg ntawm αβ-14 carotenoids. Tsis tas li ntawd, n-dodecyl β-d-maltoside (β-DDM) molecule tau daws, thiab nws tus tw hydrophobic txuas mus rau qhov chaw ntawm protein-W thiab αβ-14, thiab tus tw lipid tej zaum yuav nyob hauv lub cev. Peb kuj tau pom tias thaj chaw daws teeb meem C-terminal ntawm cov protein W thiab RCH yog ze heev, tab sis tsis nyob hauv qhov ntau thiab tsawg ntawm kev tsim cov kev sib cuam tshuam tshwj xeeb (Daim Duab 1, A thiab E). Txawm li cas los xij, tej zaum yuav muaj kev sib cuam tshuam hauv cov amino acids C-terminal uas tsis tau daws teeb meem ntawm ob cov protein no, uas yuav muab cov txheej txheem rau kev nrhiav cov protein-W thaum lub sijhawm sib sau ua ke ntawm RC-LH114-W complex.
(A) Protein-W, uas tig mus rau qhov interface nrog LH1αβ14 hauv daim duab kos duab, muaj ib sab saw hlau zoo li tus pas nrig (liab), tso tawm hauv ib feem ntawm daim duab electrostatic potential (qhov chaw grey pob tshab nrog qib contour ntawm 0.13). (B) Protein-W sawv cev los ntawm qhov chaw xim hydrophobic. Cov cheeb tsam polar thiab charged tau tso tawm hauv cyan, cov cheeb tsam hydrophobic tau tso tawm hauv dawb, thiab cov cheeb tsam hydrophobic heev tau tso tawm hauv txiv kab ntxwv. (C thiab D) Protein-W sawv cev hauv daim duab kos duab, nws qhov kev taw qhia zoo ib yam li hauv (A) (C), thiab tig los ntawm 180 ° (D). Raws li qhov chaw hauv qhov kev sib lawv liag, cov residues sib txawv txais yuav cov xim zaj sawv, qhov twg N-terminal yog xiav thiab C-terminal yog liab. (E) Protein-W nyob rau hauv tib lub zeem muag zoo li hauv (A), thiab cov residues ntawm qhov interface ntawm protein-W:LH1 yog sawv cev los ntawm cov pas nrig nrog cov cim txuas. (F) Protein-W tig 90° piv rau (E) thiab LH1αβ14 hauv daim duab kos, thiab piv rau cov seem interface hauv daim duab kab. Cov seem uas dai ntawm beta polypeptide raug cim. Tus cofactor tau pom ua ib kab sib phim xim ntawm Daim Duab 1, β-DDM uas lwj tag tau pom ua xim grey, thiab cov pa oxygen tau pom ua xim liab. (G) Daim duab hauv (F) tig 180°, nrog rau cov seem tseem ceeb ntawm alpha polypeptide uas raug cim.
Protein-W hloov αβ heterodimer (tus thib 15 hauv Daim Duab 1F), yog li tiv thaiv kev kaw lub voj voog thiab tilting thawj peb αβ heterodimers. Nws tau pom tias lub kaum sab xis siab tshaj plaws ntawm thawj αβ-1 heterodimer piv rau zaj duab xis ib txwm yog 25 ° txog 29 ° (Daim Duab 1, A thiab E), uas tau tsim los ntawm 2 ° txog 8 ° inclination ntawm αβ-1 hauv RC A ntse contrast-LH116 (Daim Duab 1, B thiab F). Qhov thib ob thiab thib peb heterodimers yog inclined ntawm 12 ° txog 22 ° thiab 5 ° txog 10 °, feem. Vim yog qhov steric hindrance ntawm RC, qhov tilt ntawm αβ-1 tsis suav nrog tus khub thib ob ntawm αβ (uas sib raug rau 16th αβ hauv Daim Duab 1F), yog li tsim qhov sib txawv meej hauv lub nplhaib LH1 (Daim Duab 1, A thiab E). Vim tsis muaj ob lub αβ heterodimers, nrog rau qhov poob ntawm plaub BChl thiab ob lub carotenoids, tsis muaj ib qho ntawm cov carotenoids khi rau lub twisted αβ-1 subunit, ua rau lub nplhaib LH114-W muaj 13 carotenoids Vegetarian thiab 28 BChls. Qhov kev kwv yees daws teeb meem hauv zos ntawm ob lub complexes hauv αβ1 txog 7 thaj tsam qis dua li cov ntawm seem ntawm LH1 lub voj voog, uas tej zaum yuav cuam tshuam qhov plasticity ntawm LH1 subunit uas nyob ib sab ntawm RC QB qhov chaw (Daim Duab 4).
Cov duab ntawm RC-LH114-W (A thiab B) thiab RC-LH116 (C thiab D) tau pom los ntawm tib lub qhov saib saum toj/sab saib (A thiab B) (A thiab C) thiab qhov chaw ntawm daim duab 1. (B thiab D). Cov yuam sij xim tau pom ntawm sab xis.
Tsuas yog lwm yam ntxwv tseem ceeb ntawm cov tub ntxhais kawm nrog qhov sib piv stoichiometric ntawm 1:14 yog Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Txawm li cas los xij, protein W thiab PufX tsis muaj qhov sib xws pom tseeb, thiab muaj qhov cuam tshuam loj rau lawv cov qauv LH1. PufX yog ib qho TMH nrog N-terminal cytoplasmic domain uas cuam tshuam nrog sab cytoplasmic ntawm RC-H subunit (13) ntawm qhov chaw sib xws rau Rps. palustris LH116αβ-16. PufX tsim ib txoj hauv kev rau kev sib pauv quinone / quinolone ntawm RC-LH1 thiab cytochrome bcl complex thiab muaj nyob hauv txhua Rba. sphaeroides core complex (13). Txawm hais tias lub monomer-monomer interface yog nyob rau hauv Rba. Tus sphaeroides RC-LH1-PufX dimer nyob ntawm qhov chaw khi ntawm cov protein W hauv RC-LH114-W, thiab qhov sib txawv uas PufX thiab protein-W tsim yog nyob rau ntawm qhov chaw sib npaug (Daim Duab S7A). Qhov sib txawv hauv RC-LH114-W kuj tseem sib phim nrog cov channel quinone hypothetical (8) ntawm Pseudomonas rosea LH1, uas yog tsim los ntawm peptides tsis muaj feem cuam tshuam nrog protein W lossis PufX (Daim Duab S7B). Tsis tas li ntawd, cov channel quinone hauv Blc. Lub LH1 ntsuab emerald tsim los ntawm kev tshem tawm ib qho γ subunit (7) nyob rau hauv qhov chaw zoo sib xws (Daim Duab S7C). Txawm hais tias mediated los ntawm cov protein sib txawv, qhov tshwm sim ntawm cov channel quinone / quinolol no hauv qhov chaw sib xws hauv RC-LH1 complex zoo li yog ib qho piv txwv ntawm kev hloov pauv sib sau ua ke, qhia tias qhov sib txawv tsim los ntawm protein W yuav ua haujlwm ua cov channel quinone.
Qhov sib txawv hauv LH114-W lub voj voog tso cai rau kev tsim ntawm thaj chaw membrane txuas ntxiv ntawm qhov chaw sab hauv ntawm RC-LH114-W complex thiab lub bulk membrane (Daim Duab 1G), es tsis txhob txuas ob lub npe los ntawm lub qhov protein zoo li hauv cov protein. Lub RC-LH116 complex zoo ib yam li lub kaw Tch. Koob zoo li lub koob (22) (Daim Duab 1H). Txij li thaum qhov kev sib kis ntawm quinone los ntawm lub membrane yog sai dua li qhov kev sib kis los ntawm cov protein channel nqaim, lub voj voog qhib LH114-W tuaj yeem tso cai rau RC tig sai dua li lub kaw LH116 lub voj voog, thiab qhov kev sib kis ntawm quinone rau hauv RC yuav raug txwv ntau dua. Txhawm rau sim seb protein W puas cuam tshuam rau kev hloov pauv ntawm quinones los ntawm RC, peb tau ua qhov kev ntsuam xyuas cytochrome oxidation ntawm qee qhov kev sib xyaw ntawm ubiquinone 2 (UQ2) (ib qho analogue ntawm ntuj UQ10 nrog tus Tsov tus tw isoprene luv dua) (Daim Duab 2E). Txawm hais tias qhov muaj chelated quinone cuam tshuam qhov kev txiav txim siab tseeb ntawm Michaelis tsis hloov pauv (RC-LH114-W thiab RC-LH116 yog qhov tsim nyog rau 0.2±0.1μM thiab 0.5±0.2μM, feem), qhov siab tshaj plaws ntawm RC-LH114-W (4.6±0.2 e-RC-1 s-1) yog 28±5% loj dua RC-LH116 (3.6±0.1 e-RC-1 s-1).
Thaum xub thawj peb kwv yees tias cov protein-W muaj nyob hauv kwv yees li 10% ntawm cov core complex (16); ntawm no, cov nqi nyob ntawm cov hlwb loj hlob uas muaj teeb pom kev tsawg, nruab nrab teeb pom kev, thiab siab teeb pom kev yog 15 ± 0.6%, 11 ± 1% thiab 0.9 ± 0.5, feem pua ​​(Daim Duab 2F). Kev sib piv ntawm cov spectrometry qhia tau hais tias kev ntxiv cov histidine tag tsis txo qhov muaj protein-W ntau piv rau cov kab mob qus (P = 0.59), yog li cov theem no tsis yog ib qho khoom cuav ntawm cov protein-W hloov kho (Daim Duab S10). Txawm li cas los xij, qhov kev nyob qis ntawm cov protein-W hauv RC-LH1 complex yuav tso cai rau qee cov RCs tig ntawm qhov nrawm, yog li txo qhov qeeb qeeb quinone / quinolone pauv hauv RC-LH116 complex. Peb pom tias qhov nqi nyob ntawm lub teeb pom kev siab tsis sib xws nrog cov ntaub ntawv transcriptomics tsis ntev los no, uas qhia tau hais tias pufW gene qhia nce ntxiv nyob rau hauv lub teeb muaj zog (Daim Duab S11) (23). Qhov sib txawv ntawm pufW transcription thiab protein-W incorporation rau hauv RC-LH1 complex yog qhov tsis meej pem thiab tej zaum yuav qhia txog kev tswj hwm ntawm cov protein.
Hauv RC-LH114-W, 6 cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) thiab 29 β-DDM molecules raug faib thiab ua qauv hauv nws 6 CDLs, 24 POPCs, 2 POPGs thiab 12 βDDMs. RC-LH116 (Daim Duab 5, A thiab B). Hauv ob lub qauv no, CDL yuav luag nyob rau sab cytoplasmic ntawm lub complex, thaum POPC, POPG thiab β-DDM feem ntau nyob rau sab luminal. Ob lub lipid thiab detergent molecules tau cais tawm hauv αβ-1 rau αβ-6 thaj tsam ntawm RC-LH114-W complex (Daim Duab 5A), thiab tsib tau cais tawm hauv thaj tsam sib npaug ntawm RC-LH116 (Daim Duab 5B). Muaj ntau cov lipids pom nyob rau sab tod ntawm lub complex, feem ntau yog CDL, sib sau ua ke ntawm RC thiab αβ-7 rau αβ-13 (Daim Duab 5, A thiab B). Lwm cov lipids thiab cov tshuaj ntxuav uas tau daws teeb meem nyob sab nraum lub nplhaib LH1, thiab cov saw hlau acyl uas tau daws teeb meem zoo txuas ntxiv ntawm LH1 subunits, sim ua β-DDM hauv RC-LH114-W, thiab txhais tias yog β-DDM hauv RC Ib qho sib xyaw ntawm β-DDM thiab POPC-LH116. Cov chaw zoo sib xws ntawm chelating lipids thiab cov tshuaj ntxuav hauv peb cov qauv qhia tias lawv yog cov chaw khi uas cuam tshuam rau lub cev (Daim Duab S12A). Cov chaw ntawm cov molecules sib npaug hauv Tch kuj muaj kev sib xws zoo. Gentle thiab Trv. Cov kab mob 970 RC-LH1s (Daim Duab S12, B txog E) (9, 12) thiab cov hydrogen-bonding residues ntawm pawg lipid taub hau tau qhia txog kev txuag zoo hauv kev sib dhos ua ke (Daim Duab S13), qhia tias Conserved CDL uas khi rau RC (24), cov chaw no yuav raug txuag hauv RC-LH1 complex.
(A thiab B) RC-LH114-W (A) thiab RC-LH116 (B) peptides yog sawv cev los ntawm cov duab tas luav, thiab cov xim yog sawv cev los ntawm cov pas nrig, siv cov xim sib txawv hauv Daim Duab 1. Cov roj ntsha tau qhia ua xim liab, thiab cov tshuaj ntxuav tau qhia ua xim grey. UQ khi rau RC QA thiab QB qhov chaw yog xim daj, thaum UQ cais yog xiav. (C thiab D) Cov kev pom zoo ib yam li (A) thiab (B), nrog cov roj ntsha tsis suav nrog. (E rau G) Kev pom loj dua ntawm Q1 (E), Q2 (F) thiab Q3 (G) los ntawm RC-LH116, nrog cov saw hlau uas cuam tshuam rau ib leeg. Cov hydrogen bonds tau qhia ua cov kab dub dashed.
Hauv RC-LH116, ob qho tib si RC QA thiab QB UQ, uas koom nrog kev hloov pauv hluav taws xob hauv cov txheej txheem sib cais them, raug rhuav tshem hauv lawv qhov chaw khi. Txawm li cas los xij, hauv RC-LH114-W, QB quinone tsis tau daws teeb meem thiab yuav raug tham txog ntau yam hauv qab no. Ntxiv rau QA thiab QB quinones, ob lub chelated UQ molecules (nyob nruab nrab ntawm RC thiab LH1 rings) raug muab faib rau hauv RC-LH114-W qauv raws li lawv cov pab pawg taub hau zoo (nyob hauv Q1 thiab Q2, feem). Qhov chaw). Daim duab 5C). Ob lub isoprene units raug muab rau Q1, thiab daim ntawv qhia qhov ceev daws qhov tiav 10 isoprene tails ntawm Q2. Hauv cov qauv ntawm RC-LH116, peb lub chelated UQ10 molecules (Q1 txog Q3, Daim duab 5D) tau daws teeb meem, thiab txhua lub molecules muaj qhov ceev meej thoob plaws hauv tus Tsov tus tw (Daim duab 5, D txog G). Hauv ob lub qauv, qhov chaw ntawm cov pawg quinone taub hau ntawm Q1 thiab Q2 muaj kev sib xws zoo heev (Daim Duab S12F), thiab lawv tsuas yog cuam tshuam nrog RC. Q1 nyob ntawm qhov nkag ntawm W qhov sib txawv ntawm RC-LH114-W (Daim Duab 1G thiab 5, C, D thiab E), thiab Q2 nyob ze ntawm qhov chaw khi QB (Daim Duab 5, C, D) thiab F). Cov seem L-Trp143 thiab L-Trp269 uas tau khaws cia yog ze heev rau Q1 thiab Q2 thiab muab kev sib cuam tshuam π-stacking (Daim Duab 5, E thiab F, thiab Daim Duab S12). L-Gln88, 3.0 Å los ntawm cov pa oxygen distal ntawm Q1, muab cov hydrogen sib txuas muaj zog (Daim Duab 5E); cov seem no tau khaws cia hauv txhua RCs tshwj tsis yog qhov kev sib raug zoo deb tshaj plaws (Daim Duab S13). L-Ser91 raug hloov pauv rau Thr hauv feem ntau lwm cov RCs (Daim Duab S13), yog 3.8 Angstroms los ntawm methyl oxygen ntawm Q1, thiab tej zaum yuav muab cov hydrogen bonds tsis muaj zog (Daim Duab 5E). Q3 zoo li tsis muaj kev sib cuam tshuam tshwj xeeb, tab sis nyob hauv thaj chaw hydrophobic ntawm RC-M subunit thiab LH1-α subunit 5 txog 6 (Daim Duab 5, D thiab G). Q1, Q2 thiab Q3 lossis cov quinones chelated nyob ze kuj tau daws teeb meem hauv Tch. Gentle, Trv. Strain 970 thiab Blc. Cov qauv iris (9, 10, 12) taw tes rau qhov chaw khi quinone auxiliary hauv RC-LH1 complex (Daim Duab S12G). Tsib lub UQs uas tau tawg ua tej daim me me hauv RC-LH116 zoo sib xws nrog 5.8 ± 0.7 ntawm txhua lub complex uas tau txiav txim los ntawm high performance liquid chromatography (HPLC), thaum peb lub UQs uas tau tawg ua tej daim me me hauv RC-LH114-W qis dua Tus nqi ntsuas ntawm 6.2 ± 0.3 (Daim Duab S14) qhia tias muaj cov UQ molecules uas tsis tau daws teeb meem hauv cov qauv.
Cov pseudo-symmetric L thiab M polypeptides txhua tus muaj tsib TMHs thiab tsim ib lub heterodimer uas muab ib qho BChl dimer, ob lub BChl monomers, ob lub bacteriophage (BPh) monomers, thiab ib qho tsis yog Heme hlau thiab ib lossis ob lub UQ10 molecules. Los ntawm qhov muaj cov hydrogen bonds ntawm pawg ketone kawg thiab nws paub tias muaj nyob rau hauv Rps, carotenoids tau koom ua ke hauv M-subunit, uas hu ua cis-3,4-dehydroorhodopin. Hom (25). Lub cheeb tsam membrane sab nraud ntawm RC-H yog anchored rau lub membrane los ntawm ib qho TMH. Tag nrho RC qauv zoo ib yam li peb lub subunit RC ntawm cov hom tsiaj txheeb ze (xws li Rba). sphaeroides (PDB ID: 3I4D). Cov macrocycles ntawm BChl thiab BPh, lub carotenoid backbone thiab cov hlau tsis yog heme sib tshooj hauv qhov kev daws teeb meem ntawm cov qauv no, ib yam li UQ10 lub taub hau pawg ntawm QA qhov chaw thiab QB quinone ntawm RC-LH116 (Daim Duab S15).
Qhov muaj ob lub qauv RC nrog cov nqi QB sib txawv ntawm qhov chaw nyob muab lub sijhawm tshiab los tshuaj xyuas cov kev hloov pauv conformational nrog rau QB quinone khi. Hauv RC-LH116 complex, QB quinone nyob rau hauv qhov chaw "proximal" tag nrho (26), tab sis qhov sib cais ntawm RC-LH114-W tsis muaj QB quinone. Tsis muaj QB quinone hauv RC-LH114-W, uas yog qhov xav tsis thoob vim tias lub complex yog active, ntau dua li RC-LH116 complex nrog cov qauv daws teeb meem QB quinone. Txawm hais tias ob lub nplhaib LH1 chelate txog rau quinones, tsib tau daws teeb meem hauv lub nplhaib RC-LH116 kaw, thaum tsuas yog peb tau txwv hauv lub nplhaib qhib RC-LH114-W. Qhov kev tsis sib haum xeeb ntawm cov qauv no tuaj yeem cuam tshuam qhov hloov pauv sai dua ntawm RC-LH114-W QB qhov chaw, quinone kinetics sai dua hauv lub complex, thiab muaj feem ntau ntawm kev hla lub voj LH1. Peb xav tias qhov tsis muaj UQ hauv RC QB qhov chaw ntawm RC-LH114-W tej zaum yuav yog qhov tshwm sim ntawm qhov nyuaj dua thiab ua haujlwm ntau dua, thiab qhov chaw QB ntawm RC-LH114-W tau khov tam sim ntawd hauv UQ turnover. Theem tshwj xeeb (qhov nkag mus rau qhov chaw QB tau raug kaw) qhia txog qhov kev hloov pauv ntawm qhov kev ua ub no.
Yog tsis muaj QB, qhov kev sib hloov ntawm L-Phe217 mus rau qhov chaw uas tsis sib haum nrog UQ10 khi, vim tias nws yuav ua rau muaj kev sib tsoo nrog thawj isoprene unit ntawm tus Tsov tus tw (Daim Duab 6A). Tsis tas li ntawd, qhov kev hloov pauv tseem ceeb pom tseeb, tshwj xeeb tshaj yog helix de (helix luv luv hauv lub voj voog ntawm TMH D thiab E) qhov twg L-Phe217 raug hloov mus rau lub hnab tshos khi QB thiab kev sib hloov ntawm L-Tyr223 (Daim Duab 6A) Txhawm rau rhuav tshem cov hydrogen bond nrog M-Asp45 lub moj khaum thiab kaw qhov nkag ntawm QB khi qhov chaw (Daim Duab 6B). Helix de pivots ntawm nws lub hauv paus, Cα ntawm L-Ser209 raug hloov los ntawm 0.33Å, thaum L-Val221Cα raug hloov los ntawm 3.52Å. Tsis muaj kev hloov pauv pom tau hauv TMH D thiab E, uas yog superimposable hauv ob qho qauv (Daim Duab 6A). Raws li peb paub, qhov no yog thawj qhov qauv hauv RC ntuj uas kaw qhov chaw QB. Kev sib piv nrog cov qauv tiav (QB-bound) qhia tau hais tias ua ntej quinone raug txo qis, kev hloov pauv conformational yog qhov yuav tsum tau ua kom nws nkag mus rau hauv quinone. L-Phe217 tig los ua ib qho kev sib cuam tshuam π-stacking nrog pawg taub hau quinone, thiab lub helix hloov mus rau sab nraud, tso cai rau lub cev pob txha ntawm L-Gly222 thiab cov saw hlau ntawm L-Tyr223 los tsim ib lub network hydrogen bond nrog cov qauv hydrogen bond ruaj khov (Daim Duab 6, A thiab C).
(A) Daim duab kos sib tshooj ntawm hologram (L saw, txiv kab ntxwv/M saw, magenta) thiab apo (grey) qauv, uas cov seem tseem ceeb tau tso tawm hauv daim ntawv ntawm tus sawv cev zoo li pas nrig. UQ10 yog sawv cev los ntawm ib txoj kab daj. Cov kab dotted qhia txog cov hydrogen bonds tsim nyob rau hauv tag nrho cov qauv. (B thiab C) Qhov sawv cev ntawm qhov chaw ntawm apolipoprotein thiab tag nrho cov qauv nplhaib, qhia txog cov saw hlau sab ntawm L-Phe217 hauv xiav thiab L-Tyr223 hauv liab, feem. L subunit yog txiv kab ntxwv; M thiab H subunits tsis muaj xim. (D thiab E) Apolipoprotein (D) thiab tag nrho (E) RC QB qhov chaw [xim los ntawm (A) feem] thiab Thermophilus thermophilus PSII (ntsuab, xiav nrog yas quinone; PDB ID: 3WU2) Align (58).
Txawm hais tias muaj ntau cov qauv ntawm QB-deficient RCs uas tsis muaj LH1, cov kev hloov pauv conformational uas tau pom hauv txoj kev tshawb fawb no tsis tau tshaj tawm ua ntej. Cov no suav nrog cov qauv QB depletion los ntawm Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) thiab Rba. sphaeroides (PDB ID: 1OGV) (29), tag nrho cov no yuav luag zoo ib yam li lawv cov qauv QB tag nrho. Kev tshuaj xyuas ze ntawm 3PRC tau qhia tias LDAO (Lauryl Dimethyl Amine Oxide) cov tshuaj ntxuav tes sib txuas ntawm qhov nkag ntawm QB txoj haujlwm, uas yuav tiv thaiv kev hloov pauv mus rau hauv qhov kaw conformation. Txawm hais tias LDAO tsis decompose ntawm tib txoj haujlwm hauv 1EYS lossis 1OGV, cov RCs no tau npaj siv tib lub tshuaj ntxuav tes thiab yog li ntawd yuav tsim cov nyhuv tib yam. Cov qauv siv lead ua ntawm Rba. Sphaeroides RC co-crystallized nrog cytochrome c2 (PDB ID: 1L9B) kuj zoo li muaj qhov chaw kaw QB. Txawm li cas los xij, hauv qhov no, thaj tsam N-terminal ntawm RC-M polypeptide (sib cuam tshuam nrog qhov chaw khi QB los ntawm H bond ntawm Tyr residue ntawm Q helix) txais yuav qhov kev hloov pauv tsis zoo li qub, thiab QB kev hloov pauv tsis tau tshawb nrhiav ntxiv (30). Qhov ua rau peb ntseeg siab yog tias peb tsis tau pom hom kev hloov pauv ntawm M polypeptide hauv RC-LH114-W qauv, uas yuav luag zoo ib yam li thaj tsam N-terminal ntawm RC-LH116 RC. Nws kuj tseem yuav tsum tau sau tseg tias tom qab kev tshem tawm ntawm lub detergent-based LH1 antenna, cov apolipoprotein RCs hauv PDB tau daws teeb meem, uas tshem tawm cov pas dej quinone sab hauv thiab lipids hauv qhov sib txawv ntawm RC thiab sab hauv ntawm lub nplhaib LH1 ib puag ncig (31, 32). RC tseem ua haujlwm vim nws khaws txhua yam cofactors, tshwj tsis yog qhov decomposable QB quinone, uas tsis ruaj khov thiab feem ntau ploj thaum lub sijhawm npaj (33). Tsis tas li ntawd, nws paub tias kev tshem tawm LH1 thiab cov roj ntsha cyclic ntuj los ntawm RC tuaj yeem muaj kev cuam tshuam rau kev ua haujlwm, xws li lub neej luv luv ntawm lub sijhawm sib cais P + QB-xeev (31, 34, 35). Yog li ntawd, peb xav tias qhov muaj nyob ntawm lub nplhaib LH1 hauv zos nyob ib puag ncig RC yuav tswj tau qhov chaw "kaw" QB, yog li khaws cia qhov chaw ib puag ncig hauv zos ze QB.
Txawm hais tias apolipoprotein (tsis muaj QB quinone) thiab cov qauv tiav tsuas yog ob daim duab ntawm qhov hloov pauv ntawm QB qhov chaw, es tsis yog ib qho xwm txheej, muaj cov cim qhia tias qhov kev khi tuaj yeem raug kaw kom tiv thaiv kev rov ua dua los ntawm hydroquinone Txhawm rau tiv thaiv kev txwv tsis pub muaj substrate. Kev sib cuam tshuam ntawm quinolol thiab quinone ze ntawm QB qhov chaw ntawm apolipoprotein tej zaum yuav txawv, uas ua rau nws tsis lees txais los ntawm RC. Nws tau ntev tau hais tias kev hloov pauv conformational ua lub luag haujlwm hauv kev khi thiab txo qis ntawm quinones. Lub peev xwm ntawm khov RCs kom txo quinones tom qab tsaus ntuj adaptation yog impaired (36); X-ray crystallography qhia tau hais tias qhov kev puas tsuaj no yog vim QB quinones raug kaw hauv "distal" conformation txog 4.5 Å los ntawm qhov chaw proximal nquag (26), 37). Peb xav tias qhov kev sib txuas distal no yog daim duab ntawm lub xeev nruab nrab ntawm apolipoprotein thiab tag nrho cov qauv nplhaib, uas ua raws li kev sib cuam tshuam thawj zaug nrog quinone thiab qhib ntawm QB qhov chaw.
Hom II RC pom nyob rau hauv PSII complex ntawm qee cov kab mob phototrophic thiab cyanobacteria, algae thiab cov nroj tsuag muaj kev txuag cov qauv thiab kev ua haujlwm (38). Kev sib dhos ua qauv qhia hauv Daim Duab 6 (D thiab E) hais txog qhov zoo sib xws ntawm PSII RCs thiab QB qhov chaw ntawm cov kab mob RC complex. Qhov kev sib piv no tau ntev los ua tus qauv rau kev kawm txog cov kab ke sib raug zoo ntawm quinone khi thiab txo qis. Cov ntawv tshaj tawm yav dhau los tau hais tias kev hloov pauv conformational yog nrog PSII txo qis ntawm quinones (39, 40). Yog li ntawd, xav txog kev txuag evolutionary ntawm RC, qhov kev khi ua ntej tsis tau pom no kuj tseem siv tau rau QB qhov chaw ntawm PSII RC hauv cov nroj tsuag oxygenated phototrophic.
Rps ΔpufW (tsis muaj daim ntawv lo pufW deletion) thiab PufW-His (C-terminal 10x His-tagged protein-W qhia los ntawm cov kab mob pufW locus ntuj). palustris CGA009 tau piav qhia hauv peb txoj haujlwm yav dhau los (16). Cov kab mob no thiab cov niam txiv isogenic qus tau rov qab los ntawm lub tub yees los ntawm kev siv cov qe me me ntawm PYE (txhua 5 g liter -1) (khaws cia rau hauv LB ntawm -80 ° C, muaj 50% (w/v) glycerol) protein, cov poov xab extract thiab succinate) agar [1.5% (w/v)] phaj. Lub phaj tau incubated hmo ntuj hauv qhov tsaus ntuj ntawm chav tsev kub hauv qab anaerobic mob, thiab tom qab ntawd ci nrog lub teeb dawb (~ 50 μmolm-2 s-1) muab los ntawm OSRAM 116-W halogen teeb (RS Components, UK) rau 3 txog 5 hnub kom txog thaum ib lub colony tshwm sim. Ib pawg kab mob tau siv los txhaj tshuaj 10 ml ntawm M22+ nruab nrab (41) ntxiv nrog 0.1% (w/v) casamino acids (tom qab no hu ua M22). Cov kab lis kev cai tau cog rau hauv qab cov pa oxygen tsawg hauv qhov tsaus ntuj ntawm 34°C nrog kev co ntawm 180 rpm rau 48 teev, thiab tom qab ntawd 70 ml ntawm cov kab lis kev cai tau txhaj tshuaj rau hauv tib qho xwm txheej rau 24 teev. Ib qho kab lis kev cai semi-aerobic nrog qhov ntim ntawm 1 ml yog siv los txhaj tshuaj 30 ml ntawm M22 nruab nrab hauv lub raj mis iav pob tshab 30 ml thiab irradiated nrog kev sib xyaw (~ 50μmolm-2 s-1) rau 48 teev los ntawm lub zog sib nqus sterile Stirring pas nrig. Tom qab ntawd 30 ml ntawm cov kab lis kev cai tau txhaj tshuaj nrog kwv yees li 1 liter ntawm cov kab lis kev cai nyob rau hauv tib qho xwm txheej, uas tom qab ntawd siv los txhaj tshuaj txog li 9 litres ntawm cov kab lis kev cai illuminated ntawm ~ 200 μmolm-2 s-1 rau 72 teev. Cov hlwb tau sau los ntawm centrifugation ntawm 7132 RCF rau 30 feeb, rov ua dua hauv ~ 10 ml ntawm 20 mM tris-HCl (pH 8.0), thiab khaws cia ntawm -20 ° C kom txog thaum xav tau.
Tom qab yaj lawm, ntxiv qee cov siv lead ua ntawm deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) thiab ob lub Roche holoenzyme protease inhibitor ntsiav tshuaj (Merck, UK) rau hauv cov hlwb uas rov ua dua tshiab. Hauv lub hlwb Fabkis 20,000 psi (Aminco, USA), cov hlwb tau raug cuam tshuam 8 txog 12 zaug. Tom qab tshem tawm cov hlwb tsis tawg thiab cov khib nyiab tsis yaj los ntawm centrifugation ntawm 18,500 RCF rau 15 feeb ntawm 4 ° C, daim nyias nyias tau precipitated los ntawm cov xim lysate los ntawm centrifugation ntawm 113,000 RCF rau 2 teev ntawm 43,000 ° C. Pov tseg cov feem uas yaj tau thiab rov ua dua daim nyias nyias xim hauv 100 txog 200 ml ntawm 20 mM tris-HCl (pH 8.0) thiab homogenize kom txog thaum tsis muaj cov aggregates pom. Lub membrane uas raug tshem tawm tau muab tso rau hauv 20 mM tris-HCl (pH 8.0) (Anatrace, USA) uas muaj 2% (w/v) β-DDM rau 1 teev hauv qhov tsaus ntuj ntawm 4°C nrog kev sib xyaw maj mam. Tom qab ntawd centrifuge ntawm 70°C kom yaj 150,000 RCF ntawm 4°C rau 1 teev kom tshem tawm cov insolubles seem.
Daim nyias nyias solubilizing los ntawm ΔpufW hom tau siv rau 50 ml DEAE Sepharose ion pauv kem nrog peb kem ntim (CV) ntawm cov khoom sib txuas [20 mM tris-HCl (pH 8.0) uas muaj 0.03% (w / v) β-DDM]. Ntxuav kem nrog ob lub CV sib txuas buffers, thiab tom qab ntawd ntxuav kem nrog ob lub khoom sib txuas buffers uas muaj 50 mM NaCl. RC-LH116 complex tau eluted nrog linear gradient ntawm 150 txog 300 mM NaCl (hauv kev sib txuas buffer) ntawm 1.75 CV, thiab cov khoom sib txuas seem tau eluted nrog cov khoom sib txuas buffer uas muaj 300 mM NaCl ntawm 0.5 CV. Sau cov spectrum absorption ntawm 250 thiab 1000 nm, khaws cov feem nrog absorbance ratio (A880/A280) ntau dua 1 ntawm 880 txog 280 nm, dilute nws ob zaug hauv binding buffer, thiab siv cov txheej txheem tib yam dua ntawm DEAE column On purification. Dilute cov feem nrog A880/A280 ratios siab dua 1.7 thiab A880/A805 ratios siab dua 3.0, ua qhov thib peb ntawm kev pauv ion, thiab khaws cov feem nrog A880/A280 ratios siab dua 2.2 thiab A880/A805 ratios siab dua 5.0. Cov tshuaj uas tau ntxuav ib feem tau muab sib xyaw ua ke rau ~ 2 ml hauv Amicon 100,000 molecular weight cut-off (MWCO) centrifugal lim (Merck, UK), thiab muab tso rau ntawm Superdex 200 16/600 qhov loj me ntawm cov kab cais tawm (GE Healthcare, US) uas muaj 200 mM NaCl buffer, thiab tom qab ntawd eluted hauv tib lub buffer ntawm 1.5 CV. Sau cov spectra nqus ntawm qhov loj me ntawm cov feem cais tawm, thiab sib xyaw cov spectra nqus nrog A880/A280 piv ntau dua 2.4 thiab A880/A805 piv ntau dua 5.8 txog 100 A880, thiab siv tam sim ntawd rau kev npaj cryo-TEM grid lossis khaws cia Khaws cia ntawm -80 ° C kom txog thaum xav tau.
Daim nyias nyias solubilizing los ntawm PufW-His hom tau siv rau 20 ml HisPrep FF Ni-NTA Sepharose kem (20 mM tris-HCl (pH 8.0) uas muaj 200 mM NaCl thiab 0.03% (w/w)) hauv IMAC buffer (GE Healthcare). v) β-DDM]. Lub kem tau ntxuav nrog tsib CVs ntawm IMAC buffer, thiab tom qab ntawd nrog tsib CVs ntawm IMAC buffer uas muaj 10 mM histidine. Lub core complex tau eluted los ntawm kem nrog tsib IMAC buffers uas muaj 100 mM histidine. Cov feem uas muaj RC-LH114-W complex yog concentrated rau ~ 10 ml hauv lub tank stirred nruab nrog Amicon 100,000 MWCO lim (Merck, UK), diluted 20 zaug nrog binding buffer, thiab tom qab ntawd ntxiv rau 25 ml Hauv DEAE Sepharose kem, plaub CVs khi rau buffer tau siv ua ntej. Ntxuav lub kem nrog plaub CV binding buffers, tom qab ntawd elute cov complex ntawm yim CVs ntawm linear gradient ntawm 0 txog 100 mM NaCl (hauv binding buffer), thiab plaub CVs seem uas muaj 100 mM binding buffer Cov residual complexes eluted ntawm sodium chloride ua ke nrog A880/A280 piv siab dua 2.4 thiab A880/A805 piv siab dua 4.6 feem tau concentrated rau ~ 2 ml hauv Amicon 100,000 MWCO centrifugal lim, thiab puv nrog 1.5 CV IMAC ua ntej Buffer equilibrated Superdex 200 16/600 qhov loj me exclusion kem, thiab tom qab ntawd eluted hauv tib lub buffer dhau 1.5 CV. Sau cov spectra nqus ntawm cov feem me me-tsis suav nrog thiab ua kom cov spectra nqus nrog A880/A280 piv dhau 2.1 thiab A880/A805 piv dhau 4.6 txog 100 A880, uas tam sim ntawd siv rau kev npaj TEM grid khov lossis khaws cia ntawm -80 ° C kom txog thaum xav tau.
Lub tshuab Leica EM GP immersion freezer tau siv los npaj cov TEM grids uas kub tsawg. Cov khoom sib xyaw tau diluted hauv IMAC buffer rau A880 ntawm 50, thiab tom qab ntawd 5μl tau thauj mus rau ntawm lub QUANTIFOIL 1.2/1.3 carbon-coated copper mesh tshiab (Agar Scientific, UK). Incubate lub grid ntawm 20 ° C thiab 60% av noo rau 30 s, tom qab ntawd blot nws qhuav rau 3 s, thiab tom qab ntawd quench nws hauv kua ethane ntawm -176 ° C.
Cov ntaub ntawv ntawm RC-LH114-W complex tau kaw rau ntawm eBIC (Electronic Bioimaging Center) (British Diamond Light Source) nrog lub tshuab microscope Titan Krios, uas ua haujlwm ntawm qhov hluav taws xob nrawm ntawm 300kV, nrog rau qhov loj me ntawm 130,000 × thiab lub zog ntawm- Xaiv qhov sib txawv ntawm 20 eV. Lub Gatan 968 GIF Quantum nrog K2 peak detector tau siv los kaw cov duab hauv hom suav kom sau cov ntaub ntawv. Qhov loj me ntawm pixel calibrated yog 1.048Å, thiab tus nqi koob tshuaj yog 3.83 e-Å-2s-1. Sau cov yeeb yaj kiab hauv 11 vib nas this thiab faib ua 40 qhov. Siv thaj chaw carbon-coated los rov tsom lub tshuab microscope, thiab tom qab ntawd sau peb cov yeeb yaj kiab ib qhov. Tag nrho, 3130 cov yeeb yaj kiab tau sau, nrog cov nqi defocus ntawm -1 thiab -3μm.
Cov ntaub ntawv rau RC-LH116 complex tau sau los ntawm kev siv lub tshuab microscope tib yam ntawm Asterbury Biostructure Laboratory (University of Leeds, UK). Cov ntaub ntawv tau sau rau hauv hom suav nrog kev loj hlob ntawm 130 k, thiab qhov loj me ntawm pixel tau calibrated rau 1.065 Å nrog koob tshuaj ntawm 4.6 e-Å-2s-1. Cov yeeb yaj kiab tau kaw cia hauv 12 vib nas this thiab muab faib ua 48 ntu. Tag nrho, 3359 zaj duab xis tau sau, nrog rau cov nqi defocus ntawm -1 thiab -3μm.
Txhua qhov kev ua cov ntaub ntawv raug ua tiav hauv Relion 3.0 pipeline (42). Siv Motioncorr 2 (43) los kho qhov kev txav ntawm lub teeb los ntawm kev ntsuas qhov hnyav, thiab tom qab ntawd siv CTFFIND 4.1 (44) los txiav txim siab qhov CTF (kev hloov pauv sib piv) parameter. Cov duab thaij duab ib txwm muaj tom qab cov theem ua tiav thawj zaug no tau pom hauv Daim Duab 2. S16. Tus qauv xaiv tsis siv neeg yog tsim los ntawm kev xaiv tes txog 250 pixels ntawm 1000 qhov me me hauv 250-pixel thav duab thiab tsis muaj kev faib tawm ob-seem (2D), yog li tsis lees txais cov kev faib tawm uas ua tau raws li cov qauv kev ua qias tuaj lossis tsis muaj cov yam ntxwv pom tseeb. Tom qab ntawd, kev xaiv tsis siv neeg tau ua tiav rau txhua daim duab me me, thiab RC-LH114-W yog 849,359 qhov me me, thiab RC-LH116 complex yog 476,547 qhov me me. Txhua yam khoom me me uas tau xaiv tau dhau los ua ob qhov kev faib tawm 2D uas tsis yog siv, thiab tom qab txhua qhov kev khiav, cov khoom me me uas ua tau raws li thaj chaw carbon, cov qauv kuaj uas muaj kuab paug, tsis muaj cov yam ntxwv pom tseeb lossis cov khoom me me uas sib tshooj heev raug tsis lees txais, ua rau muaj 772,033 (90.9%) thiab 359,678 (75.5%). Cov khoom me me raug siv rau kev faib tawm 3D ntawm RC-LH114-W thiab RC-LH116 feem. Tus qauv siv 3D thawj zaug tau tsim los ntawm kev siv txoj kev stochastic gradient descent. Siv tus qauv pib ua qhov siv, cov khoom me me uas tau xaiv tau raug faib ua plaub pawg hauv 3D. Siv tus qauv hauv pawg no ua qhov siv, ua 3D refining rau cov khoom me me hauv pawg loj tshaj plaws, tom qab ntawd siv thawj 15Å low-pass lim los npog thaj chaw solvent, ntxiv 6 pixels ntawm cov npoo mos, thiab tom qab ua tiav cov pixels los kho qhov Gatan K2 peak Modulation transfer function ntawm lub tshuab ntes sab saum toj. Rau cov ntaub ntawv RC-LH114-W, tus qauv pib no tau hloov kho los ntawm kev tshem tawm qhov ceev ntawm ntug ntawm lub ntsej muag (txiav tawm ntawm qhov ceev ntawm cov tub ntxhais hauv UCSF Chimera). Cov qauv tshwm sim (qhov kev daws teeb meem ntawm RC-LH114-W thiab RC-LH116 yog 3.91 thiab 4.16 Å, feem) yog siv ua qhov siv rau qhov thib ob ntawm kev faib tawm 3D. Cov khoom me me siv tau muab faib ua pawg hauv chav kawm 3D thawj zaug thiab tsis muaj kev sib raug zoo nrog cov zej zog. Sib tshooj lossis tsis muaj cov yam ntxwv pom tseeb. Tom qab qhov thib ob ntawm kev faib tawm 3D, pawg nrog qhov kev daws teeb meem siab tshaj plaws tau xaiv [Rau RC-LH114-W, ib pawg yog 377,703 cov khoom me me (44.5%), rau RC-LH116, muaj ob pawg, tag nrho 260,752 cov khoom me me (54.7%), Qhov twg lawv zoo ib yam tsuas yog thaum sib phim tom qab kev tig thawj zaug nrog qhov sib txawv me me]. Cov khoom me me uas xaiv tau raug rho tawm dua hauv lub thawv 400-pixel thiab ua kom zoo los ntawm 3D refining. Lub npog ntsej muag solvent yog tsim los ntawm kev siv thawj 15Å low-pass filter, 3 pixel map expansion thiab 3 pixel soft mask. Siv per-particle CTF refinement, per-particle motion correction thiab qhov thib ob ntawm per-particle CTF refinement, 3D refinement, solvent masking thiab post-processing raug ua tom qab txhua kauj ruam kom ua kom zoo dua cov qauv tshwm sim. Siv FSC (Fourier Shell Correlation Coefficient) cut-off value ntawm 0.143, qhov kev daws teeb meem ntawm cov qauv kawg ntawm RC-LH114-W thiab RC-LH116 yog 2.65 thiab 2.80Å, raws li. FSC curve ntawm cov qauv kawg tau qhia hauv Daim Duab 2. S17.
Tag nrho cov protein sequences raug rub tawm los ntawm UniProtKB: LH1-β (PufB; UniProt ID: Q6N9L5); LH1-α (PufA; UniProtID: Q6N9L4); RC-L (PufL; UniProt ID: O83005); RC-M (PufM; UniProt ID: A0A4Z7); RC-H (PuhA; UniProt ID: A0A4Z9); Protein-W (PufW; UniProt ID: Q6N1K3). SWISS-MODEL (45) tau siv los tsim ib qho qauv homology ntawm RC, uas muaj cov protein sequences ntawm RC-L, RC-M thiab RC-H thiab cov qauv siv lead ua ntawm Rba. sphaeroides RC tau siv ua ib daim ntawv qauv (PDB ID: 5LSE) (46). Siv cov cuab yeej "haum daim ntawv qhia" hauv UCSF Chimera kom haum rau tus qauv tsim rau daim ntawv qhia (47), txhim kho cov qauv protein, thiab cofactor [4 × BChl a (monomer library residue name = BCL), 2 × BPh a (BPH), ib lossis ob hom UQ10 (U10), ib qho tsis yog heme hlau (Fe) thiab ib qho 3,4-dihydrohexacarbonylcholine (QAK)] siv Coot (48) los ntxiv. Vim tias QAK tsis muaj nyob rau hauv lub tsev qiv ntawv monomer, nws tau parameterized siv cov cuab yeej eLBOW hauv PHENIX (49).
Tom ntej no, LH1 subunit tau tsim. Thaum pib, lub cuab yeej tsim kho tsis siv neeg hauv PHENIX (49) tau siv los tsim ib feem ntawm LH1 kab ke siv daim ntawv qhia thiab LH1-α thiab LH1-β protein kab ke ua cov tswv yim. Xaiv LH1 subunit tiav tshaj plaws, rho tawm nws thiab thauj nws rau hauv Coot, ntxiv cov kab ke uas ploj lawm hauv nws, thiab kho kom zoo tag nrho cov qauv ua ntej ntxiv ob BCls a (BCL) thiab spirilloxanthin (CRT) [raws li Rps qhov ceev ntawm LH1 complex thiab cov ntsiab lus carotenoid paub. Hom (17)]. Luam cov LH1 subunit tiav, thiab siv UCSF Chimera "Docking Map Tool" los dock hauv thaj chaw tsis yog qauv ntawm LH1 ceev, thiab tom qab ntawd kho nws hauv Coot; rov ua cov txheej txheem kom txog thaum txhua LH1 subunits tau raug qauv. Rau cov qauv RC-LH114-W, los ntawm kev rho tawm qhov ceev tsis tau faib hauv Coot, cov protein raug faib los ntawm cov khoom seem uas tsis yog protein hauv daim ntawv qhia USCF Chimera thiab cov cuab yeej Autobuild siv los tsim cov qauv pib, thiab cov subunits seem (protein-W) Modeling. Hauv PHENIX (49). Ntxiv cov kab ke uas ploj lawm rau cov qauv tshwm sim hauv Coot (48), thiab tom qab ntawd manually refine tag nrho subunit. Qhov ceev tsis tau faib seem haum rau kev sib xyaw ua ke ntawm lipids (PDB monomer library ID ntawm CDL = CDL, POPC = 6PL thiab POPG = PGT), β-DDM detergent (LMT) thiab UQ10 molecules (U10). Siv PHENIX optimization (49) thiab manual optimization hauv Coot (48) kom ua tiav cov qauv pib tiav kom txog thaum cov qauv txheeb cais thiab qhov zoo ntawm qhov haum tsis tuaj yeem txhim kho ntxiv. Thaum kawg, siv LocScale (50) los ua kom daim ntawv qhia hauv zos ntse, thiab tom qab ntawd ua ntau lwm lub voj voog ntawm kev ua qauv qhov ceev tsis tau faib thiab kev ua kom zoo thiab manually.
Cov peptides, cofactors thiab lwm yam lipids thiab quinones docked hauv lawv cov densities tau qhia nyob rau hauv Daim Duab 1 thiab 2. S18 txog S23. Cov ntaub ntawv txheeb cais ntawm tus qauv kawg tau qhia nyob rau hauv Rooj S1.
Tshwj tsis yog tias tau teev tseg lwm yam, cov UV/Vis/NIR absorption spectra tau sau rau ntawm Cary60 spectrophotometer (Agilent, USA) ntawm 1 nm intervals ntawm 250 nm txog 1000 nm thiab lub sijhawm sib koom ua ke ntawm 0.1s.
Dilute cov qauv hauv lub quartz cuvette nrog txoj kev 2 hli mus rau A880 ntawm 1, thiab sau cov spectrum absorption ntawm 400 thiab 1000 nm. Cov circular dichroic spectra tau sau rau ntawm Jasco 810 spectropolarimeter (Jasco, Nyiv) ntawm 1 nm intervals ntawm 400 nm thiab 950 nm ntawm tus nqi scan ntawm 20 nm min-1.
Tus coefficient ntawm molar extinction yog txiav txim siab los ntawm kev diluting lub core complex rau A880 ntawm kwv yees li 50. Dilute 10μl ntim hauv 990μl binding buffer lossis methanol, thiab sau cov spectrum absorption tam sim ntawd kom txo qis BChl degradation. Cov ntsiab lus BChl ntawm txhua tus qauv methanol tau suav los ntawm tus coefficient extinction ntawm 771 nm ntawm 54.8 mM-1 cm-1, thiab tus coefficient extinction tau txiav txim siab (51). Faib qhov ntsuas BChl concentration los ntawm 32 (RC-LH114-W) lossis 36 (RC-LH116) los txiav txim siab qhov concentration ntawm core complex, uas tom qab ntawd siv los txiav txim siab qhov absorption spectrum ntawm tib tus qauv sau hauv lub buffer Extinction coefficient. parallel. Peb qhov kev ntsuas rov ua dua tau coj mus rau txhua tus qauv, thiab qhov nruab nrab absorbance ntawm BChl Qy siab tshaj plaws tau siv rau kev suav. Tus coefficient extinction ntawm RC-LH114-W ntsuas ntawm 878 nm yog 3280 ± 140 mM-1 cm-1, thaum tus coefficient extinction ntawm RC-LH116 ntsuas ntawm 880 nm yog 3800 ± 30 mM-1 cm-1.
UQ10 raug ntsuas raws li txoj kev hauv (52). Hauv ntej, theem rov qab HPLC (RP-HPLC) tau ua tiav siv Agilent 1200 HPLC system. Yaj txog 0.02 nmol ntawm RC-LH116 lossis RC-LH114-W hauv 50μl ntawm 50:50 methanol:chloroform uas muaj 0.02% (w/v) ferric chloride, thiab txhaj cov Beckman Coulter Ultrasphere ODS 4.6 mm uas tau sib npaug ua ntej Yaj rau hauv 1 ml-1 min-1 ntawm 40°C hauv HPLC solvent (80:20 methanol:2-propanol) ntawm ib kem × 25 cm. Ua isocratic elution hauv HPLC solvent los saib xyuas absorbance ntawm 275 nm (UQ10), 450 nm (carotenoids) thiab 780 nm (BChl) rau 1 teev. Lub ncov siab tshaj plaws hauv 275 nm chromatogram ntawm 25.5 feeb tau koom ua ke, uas tsis muaj lwm yam khoom sib xyaw uas pom tau. Thaj chaw koom ua ke yog siv los xam cov molar ntawm UQ10 uas tau rho tawm nrog rau kev siv cov kab calibration uas tau xam los ntawm kev txhaj tshuaj ntawm cov qauv ntshiab los ntawm 0 txog 5.8 nmol (Daim Duab S14). Txhua tus qauv tau soj ntsuam hauv peb qhov rov ua dua, thiab qhov yuam kev tshaj tawm sib raug rau SD ntawm qhov nruab nrab.
Ib qho tshuaj uas muaj RC-LH1 complex nrog qhov siab tshaj plaws Qy nqus ntawm 0.1 tau npaj nrog 30 μM txo qis lub plawv nees cytochrome c2 (Merck, UK) thiab 0 txog 50 μMUQ2 (Merck, UK). Peb qhov qauv 1-ml tau npaj ntawm txhua qhov kev sib xyaw UQ2 thiab incubated hmo ntuj hauv qhov tsaus ntuj ntawm 4 ° C kom ntseeg tau tias kev hloov pauv tag nrho rau qhov tsaus ntuj ua ntej ntsuas. Cov tshuaj tau thauj mus rau hauv OLIS RSM1000 modular spectrophotometer nruab nrog 300 nm nplaim taws / 500 kab grating, 1.24 hli inlet, 0.12 hli nruab nrab thiab 0.6 hli qhov hluav taws xob slits. Ib lub lim dej ntev 600 nm tau muab tso rau ntawm qhov nkag ntawm cov qauv phototube thiab lub raj photomultiplier siv los tshem tawm lub teeb excitation. Qhov absorbance tau saib xyuas ntawm 550 nm nrog lub sijhawm sib koom ua ke ntawm 0.15 s. Lub teeb ci ntsa iab raug tso tawm los ntawm 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) los ntawm lub fiber optic cable ntawm 90% zog los ntawm DC2200 maub los (Thorlabs Ltd., UK) thiab raug tso tawm mus rau qhov chaw teeb ntawm lub kaum sab xis ntawm 90 ° ntawm. Lub teeb ntsuas yog tawm tsam daim iav kom rov qab txhua lub teeb uas tsis tau nqus los ntawm cov qauv. Saib xyuas qhov absorbance 10 s ua ntej lub teeb ci ntawm 50 s. Tom qab ntawd qhov absorbance tau saib xyuas ntxiv rau 60 s hauv qhov tsaus ntuj kom ntsuas qhov twg quinolol txo qis cytochrome c23 + (saib Daim Duab S8 rau cov ntaub ntawv raw).
Cov ntaub ntawv tau ua tiav los ntawm kev haum tus nqi pib linear hauv 0.5 txog 10 s (nyob ntawm qhov concentration ntawm UQ2) thiab nruab nrab cov nqi ntawm tag nrho peb cov qauv ntawm txhua qhov concentration ntawm UQ2. Qhov concentration ntawm RC-LH1 uas xam los ntawm cov coefficient extinction tau siv los hloov tus nqi mus rau hauv qhov ua haujlwm catalytic, plotted hauv Origin Pro 2019 (OriginLab, USA), thiab haum rau Michaelis-Menten qauv los txiav txim siab qhov pom tseeb Km thiab Kcat tus nqi.
Rau kev ntsuas kev nqus ib ntus, cov qauv RC-LH1 tau diluted rau ~ 2μM hauv IMAC buffer uas muaj 50 mM sodium ascorbate (Merck, USA) thiab 0.4 mM Terbutin (Merck, USA). Ascorbic acid yog siv ua tus pub electron sacrificial, thiab tert-butaclofen yog siv ua QB inhibitor kom ntseeg tau tias tus pub RC tseem ceeb tseem raug txo qis (uas yog, tsis yog photooxidized) thoob plaws hauv cov txheej txheem ntsuas. Kwv yees li 3 ml ntawm cov qauv raug ntxiv rau lub cell tig (kwv yees li 0.1 m hauv txoj kab uas hla, 350 RPM) nrog 2 hli optical txoj kev ntev kom ntseeg tau tias cov qauv hauv txoj kev laser muaj sijhawm txaus rau kev hloov pauv tsaus ntuj ntawm cov pulses excitation. Siv ~ 100-fs laser pulses los ua kom lub Ti: Sapphire laser system (Spectra Physics, USA) ua kom cov qauv zoo siab ntawm 880 nm ntawm qhov rov ua dua ntawm 1 kHz (20 nJ rau NIR lossis 100 nJ rau Vis). Ua ntej sau cov ntaub ntawv, muab cov qauv tso rau lub teeb ci ntsa iab li 30 feeb. Kev raug rau qhov no yuav ua rau QA tsis ua haujlwm (tej zaum yuav txo QA ib lossis ob zaug). Tab sis thov nco ntsoov tias cov txheej txheem no rov qab tau vim tias tom qab lub sijhawm ntev ntawm kev hloov pauv tsaus ntuj, RC yuav maj mam rov qab mus rau QA kev ua ub no. Lub Helios spectrometer (Ultrafast Systems, USA) tau siv los ntsuas cov spectra transient nrog lub sijhawm ncua ntawm -10 txog 7000 ps. Siv Surface Xplorer software (Ultrafast Systems, USA) los faib cov ntaub ntawv teeb tsa, tom qab ntawd sib koom ua ke thiab ua tus qauv. Siv CarpetView software pob (Light Conversion Ltd., Lithuania) los siv cov ntaub ntawv teeb tsa ua ke kom tau txais cov spectra sib txawv ntsig txog kev lwj, lossis siv lub luag haujlwm uas sib sau ua ke ntau lub exponents nrog cov lus teb ntawm cov cuab yeej kom haum rau qhov kev hloov pauv ntawm ib-wavelength spectral hauv Origin (OriginLab, USA).
Raws li tau hais los saum toj no (53), ib zaj duab xis photosynthetic uas muaj LH1 complex tsis muaj ob qho tib si RC thiab peripheral LH2 antenna tau npaj. Lub membrane tau diluted hauv 20 mM tris (pH 8.0) thiab tom qab ntawd thauj mus rau hauv lub quartz cuvette nrog 2 hli optical path. Ib lub 30nJ laser pulse tau siv los ua kom cov qauv ntawm 540 nm nrog lub sijhawm ncua ntawm -10 txog 7000 ps. Ua cov txheej txheem cov ntaub ntawv teeb tsa raws li tau piav qhia rau Rps. pal qauv.
Lub membrane tau pelleted los ntawm centrifugation ntawm 150,000 RCF rau 2 teev ntawm 4 ° C, thiab tom qab ntawd nws qhov absorbance ntawm 880 nm tau resuspended hauv 20 mM tris-HCl (pH 8.0) thiab 200 mM NaCl. Yaj lub membrane los ntawm kev do maj mam hauv 2% (w/v) β-DDM rau 1 teev hauv qhov tsaus ntuj ntawm 4 ° C. Cov qauv tau diluted hauv 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) rau cov protein concentration ntawm 2.5 mg ml-1 (Bio-Rad tsom xam). Kev ua ntxiv tau ua tiav los ntawm txoj kev luam tawm ua ntej (54), pib nrog kev dilution ntawm 50 μg protein rau hauv tag nrho ntawm 50 μl TEAB uas muaj 1% (w/v) sodium laurate (Merck, UK). Tom qab sonication rau 60 s, nws tau txo nrog 5 mM tris (2-carboxyethyl) phosphine (Merck, UK) ntawm 37 ° C rau 30 feeb. Rau S-alkylation, incubate cov qauv nrog 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) thiab ntxiv nws los ntawm 200 mM isopropanol Tshuag kua rau 10 feeb ntawm chav tsev kub. Proteolytic zom tau nqa tawm los ntawm kev ntxiv 2 μg trypsin / endoproteinase Lys-C sib tov (Promega UK) thiab incubated ntawm 37 ° C rau 3 teev. Lub laurate surfactant tau rho tawm los ntawm kev ntxiv 50 μl ethyl acetate thiab 10 μl 10% (v / v) LC qib trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) thiab vortexing rau 60 s. Lub sijhawm sib cais tau txhawb nqa los ntawm centrifugation ntawm 15,700 RCF rau 5 feeb. Raws li cov txheej txheem ntawm cov chaw tsim khoom, C18 spin column (Thermo Fisher Scientific, UK) tau siv los ua tib zoo nqus thiab tshem cov ntsev ntawm theem qis dua uas muaj cov peptide. Tom qab ziab los ntawm lub tshuab nqus tsev centrifugation, cov qauv tau yaj hauv 0.5% TFA thiab 3% acetonitrile, thiab 500 ng tau soj ntsuam los ntawm nanoflow RP chromatography nrog rau mass spectrometry siv cov txheej txheem system uas tau piav qhia ua ntej.
Siv MaxQuant v.1.5.3.30 (56) rau kev txheeb xyuas cov protein thiab kev ntsuas kom nrhiav tau Rps. palustris proteome database (www.uniprot.org/proteomes/UP000001426). Cov ntaub ntawv proteomics ntawm pawg neeg tau muab tso rau hauv ProteomeXchange Alliance los ntawm PRIDE tus khub repository (http://proteomecentral.proteomexchange.org) nyob rau hauv cov ntaub ntawv teev npe PXD020402.
Rau kev tshuaj xyuas los ntawm RPLC ua ke nrog electrospray ionization mass spectrometry, RC-LH1 complex tau npaj los ntawm wild-type Rps. Siv txoj kev luam tawm ua ntej (16), qhov concentration ntawm cov protein tsim tawm hauv palustris hlwb yog 2 mg ml-1 hauv 20 mM Hepes (pH 7.8), 100 mM NaCl thiab 0.03% (w/v) β- (Bio-Rad kev tshuaj xyuas)) DDM. Raws li cov txheej txheem ntawm cov chaw tsim khoom, siv 2D purification kit (GE Healthcare, USA) los rho tawm 10 μg protein los ntawm txoj kev nag lossis daus, thiab yaj cov nag lossis daus hauv 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile thiab 20% (v / v) dej. Tsib microliters tau tshuaj xyuas los ntawm RPLC (Dionex RSLC) ua ke nrog mass spectrometry (Maxis UHR-TOF, Bruker). Siv MabPac 1.2 × 100 mm kem (Thermo Fisher Scientific, UK) rau kev sib cais ntawm 60°C thiab 100μlmin -1, nrog rau qhov sib txawv ntawm 85% (v / v) cov kuab tshuaj A [0.1% (v / v) FA thiab 0.02% (V/v) TFA kua dej] rau 85% (v/v) cov kuab tshuaj B [0.1% (v/v) FA thiab 0.02% (v/v) hauv 90% (v/v) acetonitrile TFA] Siv cov qauv electrospray ionization qhov chaw thiab cov kev teeb tsa tsis siv neeg rau ntau tshaj 60 feeb, lub tshuab ntsuas qhov hnyav tau txais 100 txog 2750 m/z (qhov hnyav-rau-nqi piv). Nrog kev pab los ntawm ExPASy bioinformatics resource portal FindPept cuab yeej (https://web.expasy.org/findpept/), kos duab qhov hnyav spectrum rau cov subunits ntawm cov complex.
Cov hlwb tau loj hlob rau 72 teev nyob rau hauv 100 ml NF-qis (10μMm-2 s-1), nruab nrab (30μMm-2 s-1) lossis siab (300μMm-2 s-1) lub teeb. M22 nruab nrab (M22 nruab nrab uas ammonium sulfate raug tshem tawm thiab sodium succinate hloov los ntawm sodium acetate) hauv lub raj mis 100 ml ntsia hlau-sab saum toj (23). Hauv tsib 30-s voj voog, 0.1 micron iav hlaws dai tau hlaws ntawm qhov ntim piv ntawm 1: 1 kom lyse cov hlwb thiab txias rau ntawm dej khov rau 5 feeb. Cov khoom tsis yaj, cov hlwb tsis tawg thiab cov hlaws dai iav tau raug tshem tawm los ntawm centrifugation ntawm 16,000 RCF rau 10 feeb hauv lub rooj zaum microcentrifuge. Daim nyias nyias tau sib cais hauv Ti 70.1 rotor nrog 100,000 RCF hauv 20 mM tris-HCl (pH 8.0) nrog 40/15% (w/w) sucrose gradient rau 10 teev.
Raws li tau piav qhia hauv peb txoj haujlwm yav dhau los, kev kuaj pom His tag ntawm PufW (16). Hauv ntej, cov khoom siv purified core complex (11.8 nM) lossis daim nyias nyias uas muaj tib qho concentration ntawm RC (txiav txim siab los ntawm oxidation rho tawm qhov sib txawv spectrum thiab phim qhov load ntawm cov gel stained) hauv 2x SDS loading buffer (Merck, UK) diluted ob zaug. Cov protein tau sib cais ntawm ib qho replica 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). Ib qho gel tau stained nrog Coomassie Brilliant Blue (Bio-Rad, UK) kom thauj khoom thiab pom cov RC-L subunit. Cov protein ntawm qhov thib ob gel tau hloov mus rau methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) rau immunoassay. Lub PVDF membrane raug thaiv hauv 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 thiab 5% (w / v) mis nyuj skimmed, thiab tom qab ntawd incubated nrog anti-His thawj antibody (hauv Dilute lub antibody buffer [50 mM tris-HCl (pH 7.6), 150 mM NaCl thiab 0.05% (v / v) Tween-20] hauv 1: 1000 A190-114A, Bethyl Laboratories, USA) rau 4 teev. Tom qab ntxuav 3 zaug rau 5 feeb hauv antibody buffer, daim nyias nyias tau muab sib xyaw nrog horseradish peroxidase (Sigma-Aldrich, UK) anti-nas secondary antibody (diluted 1: 10,000 hauv antibody buffer) Incubate kom pom tseeb (5 feeb tom qab ntxuav 3 zaug hauv antibody buffer) siv WESTAR ETA C 2.0 chemiluminescence substrate (Cyanagen, Ltalis) thiab Amersham Imager 600 (GE Healthcare, UK).
Los ntawm kev kos duab qhov kev faib tawm ntawm txhua txoj kab gel lossis immunoassay, kev koom ua ke thaj chaw hauv qab lub ncov thiab xam qhov sib piv ntawm RC-L (gel) thiab Protein-W (immunoassay), hauv ImageJ (57) Ua cov duab. Cov piv no tau hloov mus rau molar piv los ntawm kev xav tias qhov sib piv ntawm RC-L rau protein-W hauv cov qauv RC-LH114-W ntshiab yog 1: 1 thiab normalizing tag nrho cov ntaub ntawv teeb tsa raws li.
Yog xav paub ntxiv txog tsab xov xwm no, thov mus saib http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
Tsab xov xwm no yog ib tsab xov xwm pub dawb uas muab faib raws li cov cai ntawm Daim Ntawv Tso Cai Creative Commons Attribution. Tsab xov xwm no tso cai rau kev siv, kev faib tawm, thiab kev tsim dua tshiab yam tsis muaj kev txwv hauv txhua qhov chaw hauv qab qhov xwm txheej uas cov haujlwm thawj tau hais kom raug.
Lus Cim: Peb tsuas thov kom koj muab koj qhov chaw nyob email kom tus neeg uas koj pom zoo rau nplooj ntawv paub tias koj xav kom lawv pom email thiab nws tsis yog spam. Peb yuav tsis ntes cov chaw nyob email twg li.
Lo lus nug no yog siv los sim seb koj puas yog tus qhua thiab tiv thaiv kev xa tawm spam tsis siv neeg.
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Cov qauv siab daws teeb meem ntawm lub teeb ntxiab 1 hauv qhov chaw tshuaj tiv thaiv muab cov kev nkag siab tshiab rau hauv cov quinone dynamics.
David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe, Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Cov qauv siab daws teeb meem ntawm lub teeb ntxiab 1 hauv qhov chaw tshuaj tiv thaiv muab cov kev nkag siab tshiab rau hauv cov quinone dynamics.
© 2021 American Association rau Advancement of Science. tag nrho cov cai. AAAS yog tus khub ntawm HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef thiab COUNTER. ScienceAdvances ISSN 2375-2548.