Sisan†Sisan ladɛrɛsi: OX11 0DE, Angletɛri, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, Angletɛri, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Reaction center light-harvesting complex 1 (RC-LH1) ye fototalan violet banakisɛw ka kɛnɛyako yɔrɔ koloma ye. An ye RC-LH1 complexe ka cryo-électron microscopie structure fila jira ka bɔ Rhodopseudomonas palustris la. RC-LH114-W complexe ka 2,65-Å resolution structure bɛ kɛ ni subunit LH1 loops 14 ye minnu bɛ RC lamini, o min bɛ tigɛ ni proteine ​​W ye, k’a sɔrɔ complexe min tɛ proteine-W ye, o ye RC composition ye pewu min lamininen don ni RC ye. A dagalen don 16 subunit LH1 loop. O jɔli ninnu sumalen bɛ hakilinaw di kinon ka fanga kan RC-LH1 complexe kɔnɔ, o la, fɛn caman b’a la ka fɛn caman Changements conformations changements (Yɛlɛma minnu ma latigɛ fɔlɔ ni kinon sirili bɛ RC QB yɔrɔ la, ka fara kinone siriyɔrɔ dɛmɛnanw sigiyɔrɔ kan, minnu bɛ dɛmɛ ka Tɛmɛ u la ka taa RC la. W poroteyini ka sigicogo kɛrɛnkɛrɛnnen bɛ LH1 jirisunba datuguli bali, o la, a bɛ sira dɔ dabɔ min bɛ kininɔn/kinɔlɔni ɲɔgɔn falen-falen teliya.
Fɛɛrɛ min bɛ sɔrɔ fotosintezi fɛ, o bɛ se ka ɲɛnamaya bɛɛ lajɛlen sabati dugukolo kan, wa a bɛ se ka kɛ tile biyoteknoloji la kosɛbɛ. K’a sɔrɔ u bɛ diɲɛ yeelenko sabatili yiriwa, fototalan wuluwuluw fana bɛ fanga cogoya ni farikoloɲɛnajɛ seko suguya caman jira. U bɛ se ka u yɛrɛ tanga yeelenbɔ ma ani ka bonya i n’a fɔ banakisɛ hɛtɛrotrofi dibi la, u bɛ se ka azote ni gazi ɲuman labɛn, ka idɔrɔzi bɔ, ka kasa dumanw tiɲɛ (1-3). Walasa ka fanga Di o wale ninnu ma, yeelen ka kan ka Yɛlɛma joona ani cogo ɲuman na ka Kɛ kemikali fanga ye. O wale in bɛ daminɛ ni yeelen-minɛn-antɛni-komplekisi ye yeelen minɛ ka fanga minɛlen bila ka taa farikoloɲɛnajɛ-yɔrɔ la (RC), o la, charge (sara) farali bɛ daminɛ (4 – 7). Fɛnɲɛnɛma-sɛnɛyɔrɔ jɔnjɔn min bɛ fototalan bulamaw la, o bɛ Kɛ ni RC suguya 2 ye, min lamininen dòn ni yeelen-sɛnɛ-yɔrɔ 1 (LH1) ye, o bɛ Kɛ RC-LH1 kɔnɔna-komplekisi ye. LH1 bɛ dilan ni αβ hɛtɛrodimɛri kurulenw ye, minnu kelen-kelen bɛɛ bɛ banakisɛ kulorofili (BChl) a mɔlikulu fila ni karotɛnoyi kelen walima fila siri (8-12). LH1 antɛni nɔgɔman bɛ kɛ ni αβ hɛtɛrodimɛri 16 walima 17 ye minnu bɛ RC (9-13) lamini jiribolo dagalen dɔ kɔnɔ, nka core complexes wɛrɛw la, transmembrane peptides bɛ LH1 lamini tɛmɛsira tigɛ , O cogo la, kinol/quinone jɛnsɛnni bɛ RC ni cytochrome bc1 complexe cɛ (11, 13-15 la). Fɛnɲɛnɛma bulama min bɛ yeelen bɔ, n’o ye Rhodopseudomonas (Rps.) ye, o ye fɛnɲɛnama misali ye min bɛ se ka fanga ni ɛlektrɔnikiw cili faamuya min bɛ fototalan dɛmɛ. Kristali jɔcogo fɔlɔ min bɛ Rps. Palustris RC-LH1 complexe modeli ye RC ye, min lamininen don ni LH1 loops hétérodimères 15 ye, minnu bɛ tigɛ ni proteine ​​dɔnbali dɔ ye min bɛ wele ko “Protéine W” (14). O kɔfɛ, poroteyini-W jirala ko RPA4402, o ye poroteyini 10,5kDa ye min tɛ taamasiyɛn ye, ni transmembrane helices (TMH) saba ye minnu fɔra ka kɔrɔ (16). An b’a ɲini ka rpa4402 jamu min bɛ poroteyini W kofɔ, o tɔgɔ caman wɛrɛ la ka kɛ pufW ye walasa a ka bɛn tɔgɔdacogo ma min bɛ kɛ jamu minnu bɛ RC-L, M (pufL, pufM) ani LH1α, β (pufA, pufB) yɔrɔ fitininw kofɔ. A nafa ka bon kosɛbɛ, proteine-W bɛ sɔrɔ RC-LH1 10% ɲɔgɔn dɔrɔn de la, o bɛ Rps jira. palustris bɛ RC-LH1 complexes fila bɔ minnu tɛ kelen ye. Yan, an bɛ cryo-EM (cryo-EM) jɔcogo caman fɔ, minnu bɛ kɛ ni core complexes fila ye, kelen bɛ ni proteine ​​W ye ani 14 αβ hétérodimères, tɔ kelen tɛ ni proteine ​​W ye ani 16 Heterodimer LH1 loop dagalen. An ka sigicogo bɛ senna-tɛgɛrɛ caman cili jira Rps ka RC-LH1 complexe faamuyali la. palustris, bawo an ye suguya kelen-kelen bɛɛ ka jamanaden kelen-kelen bɛɛ sɛgɛsɛgɛ ani ka ɲɛfɔli bɛrɛ sɔrɔ walasa ka pepitiri kelen-kelen bɛɛ ni ɲɛgɛn sirilenw ni lipidi ni kinɔn minnu bɛ tali kɛ ɲɔgɔn na, olu di ɲɔgɔn ma ka jɛya. Nin sigida ninnu sumalen b’a jira ko TMH poroteyini-W saba minnu ma sɔrɔ RC-LH1 complexe wɛrɛ la fɔlɔ, olu bɛ kinɔni sira dɔ lawuli walasa ka kinɔni/kinɔni ɲɔgɔn falen-falen teliya. Lipidi ni kinɔn siriyɔrɔ damadɔ minnu maralen don, olu dɔnna, wa an ye fɛn kura dɔ jira min bɛ fɛn caman Changement conformational, quinone ni RC faralen ɲɔgɔn kan, o bɛ se ka bɛn fotositɛmu II (PSII) RC ma, oksizɛni bɛ fototalan fɛnɲɛnamaw la. An ka sɔrɔw bɛ hakilina kuraw di kinɔni/kinɔlɔni sirili ni ɲɔgɔn falen-falencogo kan RC-LH1 core complexe kɔnɔ, min ye fototalan wuluwuluw ye.
Walasa ka sɛgɛsɛgɛli caman nɔgɔya complexes fila minnu bɛ sɔrɔ Rps. palustris, an bɛ RC-LH1 kelen-kelen bɛɛ bɔ ɲɔgɔn na ni biyokimiki fɛɛrɛw ye. Protéine W dɛsɛlen complexe (a bɛ fɔ a ma kɔfɛ ko ΔpufW) saniyalen don ka bɔ suguya la min tɛ pufW jamu sɔrɔ (16), wa RC-LH1 complexe kelen dɔrɔn de bɛ se ka dilan. Protéine W bɛ sɔrɔ complexe min na, o bɛ dilan ni suguya dɔ ye. Nin suguya in ka poroteyini W bɛ ladilan ni 10x His taamasiyɛn ye a C-terminus la, walasa poroteyini W bɛ min na, o ka se ka fara ɲɔgɔn kan ka ɲɛ ni poroteyini W dɛsɛlen fanba ye ni nɛgɛ jɔli ye. O complexe bɛ danfara ka ɲɛ (16) Affinity Chromatography (IMAC).
I n’a fɔ a jiralen bɛ cogo min na ja 1 na, o complexe fila bɛɛ kɔnɔ, RC sub-unit saba (RC-L, RC-M ani RC-H) bɛ yen minnu lamininen don ni LH1 antenne ye. 2,80-A jɔcogo min bɛ kɛ ni poroteyini-W tɛ min na, o bɛ αβ hɛtɛrodimɛri 16 jira, minnu bɛ LH1 jiribolo dagalen dɔ dilan min bɛ RC lamini pewu, n’a bɛ fɔ o ma kɔfɛ ko RC-LH116 ɲɔgɔndan. Protéine-W bɛ min na, o 2,65Å jɔcogo bɛ ni 14-hétérodimère LH1 ye min tigɛra ni proteine-W ye, o bɛ wele kɔfɛ ko RC-LH114-W.
(A ani B) Ɲɛgɛnɛsiraw jiracogo min bɛ kɛ ni fɛnɲɛnamafagalan ye. (C ani D) Pigments sirilenw minnu bɛ jira berew la. (E ni F) Komplekisi minnu kɔlɔsira ka bɔ sitoplasma sanfɛla la, olu bɛ ni pepitiriw ni LH1 yɔrɔ fitininw ye minnu jiralen don dessin animéw kɔnɔ, wa u jate bɛ kɛ waatibolodacogo la ka bɔ poroteyini ni W cɛ [o bɛ bɛn Rba nimɔrɔ ma. sphaeroides complexe (13)) ye. LH1-α ta la, farikolojɔli dumuniw yɔrɔ kulɛri ye jɛman ye; LH1-β ta la, farikolojɔli dumuniw yɔrɔ kulɛri ye bulu ye; ka ɲɛsin poroteyini-W ma, farikolojɔlifɛn ye bilen ye; RC-H ta la, a ye siyan ye; RC-L ta la, o ye Orange ye; ka ɲɛsin RC-M ma, o ye magenta ye. Kofaktorw bɛ jira ni berew ye, wuluwulu bɛ BChl ni BPh a mɔlikuluw jira, bulama bɛ karotɛnoyiw jira, ani jɛman bɛ UQ10 mɔlikuluw jira. (G ni H) Protéine-W danfara filɛli bonyalen RC-LH114-W complexe (G) ni RC-LH116 complexe (H) yɔrɔ bɛnnin na. Cofactors bɛ jira yɔrɔ facogo la, kinone chelated bɛ jira ni bulu ye. Protéine-W danfara bɛ jira ni tigɛli bulu ye (G) kɔnɔ, ani dingɛ misɛnnin minnu na kinone/quinolol bɛ jɛnsɛn LH116 bololanɛgɛ kan, olu bɛ jira ni tigɛli nɛrɛma ye (H) kɔnɔ.
Jaa 1 (A ani B) bɛ RC jira min lamininen don ni LH1αβ hɛtɛrodimɛriw ye minnu dabɔlen don walima minnu datugulen don, minnu kelen-kelen bɛɛ bɛ BChl fila ni karotɛnoyi kelen siri (Jaa 1, C ani D). Kalan tɛmɛnenw y’a jira ko Rps ye LH1 complexe ye. Spirulina xanthin ka biyosintɛzi sira la, o suguya ninnu na, karotɛni jamana ɲagaminenw bɛ sɔrɔ (17). Nka, spiropyrroxanthin ye karotɛni ye min ka ca ni tɔw bɛɛ ye wa a hakɛ bɛ mɔgɔ wasa. O la, an y’a sugandi ka spiroxanthin modeli LH1 siriyɔrɔw bɛɛ la. Alfa ni beta polipɛtidiw ye TMH kelenw ye minnu kɔkanna yɔrɔw ka surun (Jaa 1, A, B, E, ani F). Hali ni tolen 17 density ma Kɔlɔsi C-terminus la, alfa polipɛtidi tigɛra ka bɔ Met1 la ka taa Ala46 la complexes fila bɛɛ la. β polipɛtidi dɔgɔyara ka bɔ Gly4 la ka kɛ Tyr52 ye RC-LH116 la, ani ka bɔ Ser5 la ka kɛ Tyr52 ye RC-LH114-W la. N-terminal 3 walima 4 walima C-terminal tolenw 13 density si ma ye (Ja S1). RC-LH1 complexe ɲagaminen min labɛnna ka bɔ kungo suguya la, o sɛgɛsɛgɛli kɛra mass spectrometry kan, o y’a jira ko yɔrɔ min tununna, o kɛra o pepitiri ninnu ka hɛtɛrolozi sɔgɔli de nɔ ye (Ja S1 ani S2). α-Met1 ka N-terminal formylation fana kɔlɔsira (f). Sɛgɛsɛgɛli y’a jira ko α-peptide bɛ kɛ ni tolenw ye fMet1 ka se Asp42/Ala46/Ala47/Ala50 ma, ani β-peptide bɛ kɛ tolenw ye Ser2 ka se Ala53 ma, o bɛ bɛn kosɛbɛ ni EM density map ye min bɛ funteni dɔgɔya.
α-His29 ni β-His36 ka jɛɲɔgɔnya bɛ BChls kɛ ɲɛda ni ɲɛda; αβ hɛtɛrodimɛri kelen-kelen bɛɛ bɛ ɲɔgɔn sɔrɔ n’a sigiɲɔgɔnw ye walasa ka dakun dafalen dɔ (RC-LH114-W) walima jiribolo dafalen dɔ (RC-LH116) kɛ ka RC lamini The exciton coupled pigment array (Ja 1, C ani D). Ni an y’a suma ni RC-LH114-W ka 877 nm band ye, RC-LH116 ka 880 nm absorption red shift ye 3 nm ye (Ja 2A). Nka, circular dichroism spectrum bɛ ɲini ka kɛ kelen ye (Ja 2B), o b’a jira ko hali ni danfara jɛlen bɛ dakun dafalenw ni dakun datugulenw cɛ, BChls sigida lamini bɛ tali kɛ ɲɔgɔn na kosɛbɛ. Absorption redshift bɛ se ka kɛ funteni lamagacogo dɔgɔyali ni sabatili cayalen ye jiribolo datugulen kan (18, 19), ɲɛgɛnɛsira datugulen yeli yeli fɛ (20, 21), walima nin nɔ fila ninnu faralen ɲɔgɔn kan (11).
(A) Ultraviolet/visible/near-infrarouge absorption spectrum, minnu kuncɛw bɛ taamasiyɛn kɛ n’u ɲɛkisɛw ye minnu bɛ bɛn u ma, wa u bɛ normalize ka se BPh kuncɛ ma 775 nm la. (B) Spectre dichroisme circulaire normalisé ka BChl absorbance kɛ 805 nm la. (C ani D) ΔA spekter sugandilenw ka bɔ RC-LH114-W complexe (C) ani RC-LH116 complexe (D) ka absorption spectres waati latigɛlenw na. Walasa ka ɲɔgɔn sanga ka ɲɛ, spekter bɛɛ bɛ normalize ka kɛ ∆A ye −A la 0,2 ps la. (E) Sitokɔrɔmu c2 oksidan hakɛ fiyɛli kɔfɛ UQ2 hakɛ suguya caman sɔrɔli la (aw ye ja S8 lajɛ walasa ka kunnafoni raw sɔrɔ). . Aw bɛ farikolojɔli dumuni hakɛ dɔn ni SDS-polyacrylamide gel electrophoresis ni immunoassay ye (aw ye ja S9 lajɛ walasa ka kunnafoni raw sɔrɔ). Aw bɛ hakɛ jateminɛ ka kɛɲɛ ni RC-LH114-W complexe saniyalen ye. RC-L ni proteine-W hakɛ stoikiometriki hakɛ ye 1:1 ye.
BChls minnu bɛ yɔrɔ 1 na RC-LH114-W ka αβ14 loop deformé kɔnɔ (Ja 1, A, C, ani E) olu ka surun RC donna fɔlɔ (P) la ni 6,8Å ye ka tɛmɛ BChls bɔlenw kan RC-LH116 kɔnɔ (Ja 1, B, D, ani F, ani Jaa S3) nka, o ɲɔgɔndan fila ninnu ka waati tɛmɛnenw ka minɛcogo b’a jira ko RC-LH114-W ni RC-LH116 ta la, wuli fanga jiginni waati basigilenw ka bɔ LH1 la ka taa RC la, olu ye 40 ±4 ani 44±3 ps ye (Jaa 2). , C ni D, Jaa S4 ani Tablo S2). Danfaraba fana tɛ ɛntɛrinɛti kan RC kɔnɔ (Ja S5 ani sɛbɛn faralen ɲɔgɔn kan). An bɛ sigasiga ko fanga jiginni waati bɛnkan gɛlɛn min bɛ LH1 ni RC-P cɛ, o sababu bɛ bɔ BChl fanba ka yɔrɔjan, u kɛrɛ ani fanga seko ni dɔnko ɲɔgɔn na LH1 jirisun fila kɔnɔ. A bɛ iko LH1 fanga cogoya sɛgɛsɛgɛli walasa ka se yɔrɔjan fitinin ma, o tɛ teliya ka tɛmɛ fanga jiginni tilennen kan ka bɔ yɔrɔw la minnu man ɲi ka taa RC la. LH1 dakun dafalen min bɛ RC-LH114-W kɔnɔ, o fana bɛ se ka kɛ funteni lamagacogo nafama ye funteni dɔgɔyalen cogoyaw la walasa ka sigicogo sɛgɛsɛgɛli kɛ, wa αβ14 bololanɛgɛ cogoya janyalen bɛ so funteni na ka bɔ βBChls ka pigmentation yɔrɔjan na RC 1 yɔrɔ la.
RC-LH116 complexe kɔnɔ, BChls 32 ani carotenoïdes 16 bɛ sɔrɔ, wa a labɛncogo bɛɛ lajɛlen ni min sɔrɔla Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 suguya ( PDB ID 7C9R) (12) ani green fɛ, o ye kelen ye jirisunw (Blc.viridis) (PDB ID 6ET5) (10). Labɛnni kɔfɛ, fɛn misɛnninw dɔrɔn de kɔlɔsira αβ hɛtɛrodimɛriw jɔyɔrɔw la, kɛrɛnkɛrɛnnenya la 1-5, 15 ani 16 (Jaa S6). Protéine-W sɔrɔli bɛ nɔba bila LH1 jɔcogo la. A ka TMH saba bɛ ɲɔgɔn cɛ ni jiribolo surunw ye, N-terminal bɛ complexe lumen fan fɛ ani C-terminal bɛ cytoplasmic fan fɛ (Ja 1A ni 3, A ka taa D la). Protéine-W ka ca ni ji ye (Jaa 3B), wa TMH2 ni TMH3 bɛ ɲɔgɔn sɔrɔ ni LH1αβ-14 ye ka kɛ transmembrane surface ye (Jaa 3, B ani E ka taa G la). O ɲɔgɔndan in bɛ Kɛ kosɛbɛ Phe, Leu ani Val tolenw ye minnu bɛ transmembrane (kɔnɔna) yɔrɔ la. O tolenw bɛ dalajɛ ni asidi aminiki jilamaw ni αβ-14 ɲɛgɛnw ye. Polari tolen dɔw fana bɛ kɛ sababu ye ka jɛɲɔgɔnya in kɛ, i n’a fɔ idɔrɔzi jɛɲɔgɔnya min bɛ W-Thr68 ni β-Trp42 cɛ, o min bɛ kɛ sababu ye ka yɔrɔ gɛlɛn in sanfɛ (Jaa 3, F ani G). Sitoplasma sanfɛ, Gln34 bɛ αβ-14 karotɛnoyiw ka keto kulu kɛrɛfɛ. Ka fara o kan, n-dodecyl β-d-maltoside (β-DDM) molekilɛri ɲɛnabɔra, wa a ji-sɔrɔ-kɔnɔna janyalen don fo ka se poroteyini-W ni αβ-14 cɛsira ma, wa tulumafɛnw kɔ bɛ se ka sɔrɔ farikolo la. An y’a kɔlɔsi fana ko poroteyini W ni RCH ka C-terminal resolution yɔrɔw bɛ gɛrɛ ɲɔgɔn na kosɛbɛ, nka u tɛ jɛɲɔgɔnya kɛrɛnkɛrɛnnenw dilancogo kɔnɔ (Jaa 1, A ani E). Nka, jɛɲɔgɔnya bɛ se ka kɛ nin farikolojɔlan fila ninnu ka C-terminal amino acids (amino acides) ɲɛnabɔbaliw la, o bɛ se ka kɛ sababu ye ka fɛɛrɛ dɔ di proteine-W sɔrɔli ma RC-LH114-W complexe lajɛ waati.
. (B) Protéine-W min jiralen don ni yɔrɔ kulɛrilen ye min tɛ ji bɔ. Polar ni charged yɔrɔw bɛ jira ni siyan ye, ji bɛ yɔrɔ minnu na, olu bɛ jira ni fini jɛman ye, ani jibɔyɔrɔ barikamaw bɛ jira ni orange ye. (C ni D) Protéine-W jiralen don dessin animé kɔnɔ, a ɲɛsincogo ni (A) (C) ta ye kelen ye, wa a bɛ wuli ni 180° ye (D). Ka Kɛɲɛ ni jɔyɔrɔ ye ɲɔgɔndan na, tolen minnu bɛ Se ka Bɔ ɲɔgɔn na olu bɛ sanji-kɛnɛ kulɛri ta, N-dakun ye bulu ye, C-dakun ye bilen ye. (E) Protéine-W yecogo kelen na i n’a fɔ (A), ani tolenw minnu bɛ proteine-W:LH1 cɛtigɛyɔrɔ la, olu jiralen don ni berew ye minnu taamasiyɛnw nɔrɔlen don. (F) Protéine-W bɛ wuli 90° ka kɛɲɛ ni (E) ni LH1αβ14 ye dessin animé jiracogo la, ani ka kɛɲɛ ni interface tolenw ye bar jiracogo la. Tolen minnu bɛ wuli ka bɔ beta polipɛtidi la, olu bɛ taamasiyɛn kɛ. Kofactor jiralen don i n’a fɔ bara min bɛ bɛn ja 1 kulɛri ma, β-DDM tolilen jiralen don ni nɛrɛmuguma ye, ani oksizɛni jiralen don ni bilen ye. (G) Ja min bɛ (F) kɔnɔ, o bɛ wuli 180°, ni alfa polipɛtidi taamasiyɛnlen tolenw ye minnu bɛ ye.
Protéine-W bɛ αβ hɛtɛrodimɛri dɔ bila a nɔ na (a 15nan ja 1F kɔnɔ), o la, a bɛ lupu da tugu ani ka αβ hɛtɛrodimɛri saba fɔlɔw kɔrɔta. A kɔlɔsira ko αβ-1 hɛtɛrodimɛri fɔlɔ ka jiginni yɔrɔ min ka bon ni filimu normal ye, o ye 25° fo 29° ye (Ja 1, A ani E), o min kɛra αβ-1 jiginni fɛ 2° ka se 8° ma RC A sharp contrast-LH116 kɔnɔ (Jaa 1, B ani F). Hɛtɛrodimɛri filanan ni sabanan bɛ Jigin 12° fo 22° ani 5° fo 10°. K’a sababu kɛ RC ka stɛriki balili ye, αβ-1 kɔrɔtalen tɛ αβ fila filanan sen don a la (o bɛ bɛn αβ 16nan ma ja 1F kɔnɔ), o la, a bɛ danfara jɛlen kɛ LH1 bololanɛgɛ kɔnɔ (Ja 1, A ani E) K’a sababu kɛ αβ hɛtɛrodimɛri fila dɛsɛ ye, ka fara BChl naani ni karotɛnoyi fila bɔnɛni kan, karotɛnoyi si tɛ siri αβ-1 yɔrɔ tigɛlen na, o bɛ na ni LH114-W bololanɛgɛ ye min kɔnɔ karotɛnoyi 13 bɛ sɔrɔ Nakɔfɛnw na ani BChl 28. αβ1 fo 7 maraw kɔnɔ, fɛnɲɛnamafagalan fila ninnu ka sigida ɲɛnabɔli jateminɛw ka dɔgɔ ni LH1 jirisun tɔw ta ye, o bɛ se ka LH1 yɔrɔ fitinin ka plastiki danma jira min bɛ RC QB yɔrɔ kɛrɛfɛ (Ja 4)
RC-LH114-W (A ni B) ni RC-LH116 (C ni D) jaw jiralen don ka bɔ sanfɛla lajɛ/kɛrɛfɛla lajɛ kelen na (A ni B) (A ni C) ani ja 1. (B ni D) kɔnɔna na. Kulɛriw bɛ jira kinin fɛ.
O kelenpe min bɛ danfara don a kɔnɔ, n’a ka stoikiometri hakɛ ye 1:14 ye, o ye Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer ye (13). Nka, poroteyini W ni PufX tɛ ni ɲɔgɔndɛmɛ jɛlen ye, wa u bɛ nɔba bila u kelen-kelen bɛɛ ka LH1 jɔcogo la. PufX ye TMH kelen ye min bɛ ni N-terminal cytoplasmic domain ye min bɛ baara kɛ ni RC-H subunit (13) sitoplasmic fan ye yɔrɔ la min bɛ bɛn Rps ma. palustris LH116αβ-16 ye a ka baara ye. PufX bɛ sira dɔ da kinɔni/kinɔlɔni ɲɔgɔn falen-falen kama RC-LH1 ni sitokɔrɔmu bcl kɔmplɛsi cɛ ani a bɛ sɔrɔ Rba bɛɛ la. sphaeroides core complex (13) ye. Hali ni monomɛri ni monomɛri cɛsira bɛ Rba kɔnɔ. Sphaeroides RC-LH1-PufX dimɛri bɛ sɔrɔ poroteyini W siriyɔrɔ la RC-LH114-W kɔnɔ, ani PufX ni poroteyini-W bɛ danfara min lase, o bɛ yɔrɔ kelen na (Jaa S7A). RC-LH114-W kɔnɔ danfara fana bɛ bɛn Pseudomonas rosea LH1 ka kinɔn kanal (8) hakilinata ma, o min bɛ sɔrɔ pepitiriw fɛ minnu ni poroteyini W walima PufX tɛ kelen ye (Ja S7B). Ka fara o kan, kinone kanal min bɛ Blc. LH1 jɛman emɛridi min bɛ sɔrɔ ni γ yɔrɔ kelen (7) bɔli ye, o bɛ sɔrɔ o cogo kelen na (Jaa S7C). Hali ni a bɛ kɛ sababu ye ni farikolojɔlifɛn suguya wɛrɛw ye, nin kinɔn/kinololi sira ninnu bɔli yɔrɔ kelen na RC-LH1 complexe kɔnɔ, a bɛ iko o ye sɛgɛsɛgɛli ye min bɛ kɛ ka ɲɔgɔn sɔrɔ fɛnɲɛnamafagalanw na, o b’a jira ko danfara min bɛ sɔrɔ farikolojɔlifɛn W fɛ, o bɛ se ka kɛ kinɔni sira ye.
LH114-W bɔgɔlan kɔnɔ danfara bɛ sira Di RC-LH114-W complexe kɔnɔna yɔrɔ ni bulk membrane cɛ (Ja 1G), sanni ka yɔrɔ fila ninnu siri ɲɔgɔn na poroteyini pore fɛ i n’a fɔ a bɛ kɛ cogo min na poroteyini kɔnɔ. RC-LH116 complexe bɛ tali kɛ Tch dagalen na. Komplekisi min bɛ i n’a fɔ pikirijikɛlan (22) (Jaa 1H). Ikomi kinɔni jɛnsɛnni membrane fɛ ka teliya ka tɛmɛ jɛnsɛnni kan poroteyini sira fitinin fɛ, LH114-W jirisun dafalen bɛ se ka sira di RC jiginni teliya ma ka tɛmɛ LH116 jirisun datugulen kan, wa kinɔn jɛnsɛnni RC kɔnɔ, o bɛ se ka dan ka tɛmɛ. Walasa k’a lajɛ ni poroteyini W bɛ nɔ bila kinɔnw jiginni na RC fɛ, an ye sitokɔrɔmu oksidan sɛgɛsɛgɛli kɛ ubikinɔni 2 (UQ2) hakɛ dɔ kan (UQ10 nafama analozi min ka isopɛrɛni kɔ ka surun) (Jaa 2E). Hali ni kinon kelalen sɔrɔli bɛ Michaelis constant yelen dɔnni tiɲɛ (RC-LH114-W ni RC-LH116 bɛnnen don 0,2±0,1μM ani 0,5±0,2μM ma), RC-LH114-W hakɛ cayalenba ( 4,6±0,2 e-RC-1 s-1) ye 28±5% ka bon ni RC-LH116 ye (3,6±0,1 e-RC-1 s-1).
An y’a jateminɛ fɔlɔ ko proteine-W bɛ sɔrɔ 10% ɲɔgɔn na core complex (16); yan, yeelen dɔgɔman, yeelen cɛmancɛ, ani yeelenba falen seliluw sigiyɔrɔ hakɛ ye 15±0,6%, 11±1% ani 0,9±0,5 ye, o cogo kelen na % (Jaa 2F). Mas spectrometry (mass spectrometry) hakɛ sumalen y’a jira ko histidine tag farali ma dɔ bɔ poroteyini-W caya danfaralen na ni i y’a suma ni kungo kɔnɔ suguyaw ye (P = 0,59), o la nin hakɛ ninnu tɛ poroteyini-W caman sɛmɛntiyalenw ka fɛn dilannen ye (Ja S10). Nka, nin poroteyini-W minɛni dɔgɔyalen in RC-LH1 complexe kɔnɔ, o bɛ se ka kɛ sababu ye ka RC dɔw wuli ka wuli teliya la, o bɛ kɛ sababu ye ka kinone/quinolone ɲɔgɔn falen-falen dɔɔnin dɔɔnin nɔgɔya RC-LH116 complexe kɔnɔ. An y’a kɔlɔsi ko yeelen minɛni hakɛ cayalenba tɛ bɛn kɔsa in na transcriptomics kunnafoniw ma, o b’a jira ko pufW jamu jirali bɛ bonya yeelen barikama kɔrɔ (Ja S11) (23). Danfara min bɛ pufW sɛbɛnni ni poroteyini-W doncogo cɛ RC-LH1 complexe kɔnɔ, o bɛ mɔgɔ hakili ɲagami, wa a bɛ se ka protéine ka complexe regulation jira.
RC-LH114-W kɔnɔ, karidiyolipini (CDL) 6, fosfatidilikolini (POPC) 7, fosfatidiligilisɛrɔli (POPG) 1 ani β-DDM mɔlikulu 29 bɛ tila ka kɛ misali ye a kɔnɔ CDL 6, POPC 24, POPG 2 ani βDDM 12. RC-LH116 (Jaa 5, A ani B). Nin jɔcogo fila in na, CDL bɛ ɲini ka sɔrɔ complexe (komplekisi) sitoplasma fan fɛ, k’a sɔrɔ POPC, POPG ani β-DDM fanba bɛ sɔrɔ luminal fan fɛ. Lipidi ni safinɛ mɔlikulu fila bɔra RC-LH114-W complexe αβ-1 ka se αβ-6 yɔrɔ la (Ja 5A), ani duuru bɔra RC-LH116 yɔrɔ la min bɛ bɛn o ma (Ja 5B ). Lipidi caman sɔrɔla complexe fan dɔ fɛ, kɛrɛnkɛrɛnnenya la CDL, minnu dalajɛlen don RC ni αβ-7 cɛ ka se αβ-13 ma (Jaa 5, A ani B). Lipidi ni safinɛ wɛrɛw minnu labɛnna cogo la, olu bɛ LH1 bololanɛgɛ kɔkan, wa asili cakɛda minnu ɲɛnabɔlen don koɲuman, olu bɛ janya LH1 yɔrɔ fitininw ni ɲɔgɔn cɛ, minnu bɛ fɔ a daminɛ na ko β-DDM RC-LH114-W kɔnɔ, wa u ɲɛfɔlen don ko β-DDM RC kɔnɔ β-DDM ni POPC-LH116 ɲagaminen. Chelating lipids ni detergents jɔyɔrɔ kelen minnu bɛ an ka sigida la, olu b’a jira ko u ye farikolo siriyɔrɔw ye minnu bɛ tali kɛ farikolo la (Jaa S12A). Molecules (molecules) minnu bɛ Bɛn ɲɔgɔn ma olu jɔyɔrɔw fana bɛ Blà ɲɔgɔn na kosɛbɛ. Dususuma ni Trv. RC-LH1 suguya 970 (Ja S12, B ka taa E) (9, 12) ani lipid kunkolo kulu ka hidrɔzɛni sirili tolenw ye lakanani ɲuman jira kosɛbɛ ɲɔgɔndan sinsinni na (Ja S13), o b’a jira ko CDL maralen min bɛ siri RC la (24), o yɔrɔw bɛ se ka mara RC-LH1 complexe kɔnɔ.
(A ni B) RC-LH114-W (A) ani RC-LH116 (B) pepitiriw jiralen don ni dessin animéw ye, ani ɲɛgɛnw jiralen don ni berew ye, ka baara kɛ ni kulɛriw ye minnu bɛ ja 1 kɔnɔ. UQ min sirilen bɛ RC QA ni QB yɔrɔw la, o ye jɛman ye, k’a sɔrɔ UQ min bɛ danfara la, o ye bulu ye. (C ni D) O ye hakilina kelenw ye ni (A) ni (B) ye, ni tulumafɛnw bɔra. (E ka taa G) Q1(E), Q2(F) ani Q3(G) yecogo bonyalen ka bɔ RC-LH116 la, ni kɛrɛfɛ cakɛdaw ye minnu bɛ nɔ bila ɲɔgɔn na. Hidrɔzɛni jɛɲɔgɔnyaw jiralen don i n’a fɔ tigɛli nɛrɛma.
RC-LH116 kɔnɔ, RC QA ni QB UQ fila bɛɛ, minnu bɛ u sen don ɛlektrɔnikiw tali la charge (sara) danfara la, olu bɛ tiɲɛ u siriyɔrɔw la. Nka, RC-LH114-W kɔnɔ, QB kinone ma ɲɛnabɔ, wa a bɛna ɲɛfɔ ka ɲɛ jukɔrɔ. Ka fara QA ni QB kinɔnw kan, UQ mɔlikulu fila minnu bɛ kela (minnu bɛ RC ni LH1 bololanɛgɛw cɛ) olu bɛ tila RC-LH114-W sigicogo la ka kɛɲɛ n’u kunkolo kuluw ye minnu ɲɛnabɔra ka ɲɛ (minnu bɛ Q1 ni Q2 kɔnɔ, olu kelen-kelen bɛɛ). yɔ́rɔ). Jaa 5C). Isoprene unit fila bɛ di Q1 ma, ani density map bɛ Q2 isoprene 10 dafalenw ɲɛnabɔ. RC-LH116 jɔcogo la, UQ10 fɛnɲɛnamafagalan saba (Q1 ka se Q3 ma, ja 5D) ɲɛnabɔra, wa fɛnɲɛnamafagalan bɛɛ bɛ ni density jɛlen ye u kɔ bɛɛ la (Jaa 5, D ka taa G la). O jɔcogo fila in na, Q1 ni Q2 kinin kunkolo kuluw jɔyɔrɔw bɛ bɛn ɲɔgɔn ma kosɛbɛ (Ja S12F), wa u bɛ ɲɔgɔn sɔrɔ ni RC dɔrɔn de ye. Q1 bɛ RC-LH114-W ka W gap da la (Ja 1G ni 5, C, D ani E), ani Q2 bɛ QB siriyɔrɔ kɛrɛfɛ (Ja 5, C, D) ani F). L-Trp143 ni L-Trp269 tolenw maralenw bɛ surunya kosɛbɛ Q1 ni Q2 la, wa u bɛ π-stacking jɛɲɔgɔnyaw di minnu bɛ se ka kɛ (Ja 5, E ani F, ani Jaa S12). L-Gln88, 3,0 Å ka bɔ Q1 ka oksizɛni yɔrɔjan na, o bɛ hidrɔzɛni jɛɲɔgɔnya barikama di (Jaa 5E); o tolen in bɛ mara RC bɛɛ la fo jɛɲɔgɔnya min ka jan kosɛbɛ (Jaa S13). L-Ser91 bɛ Thr nɔnabila cogo kɔrɔlen na RC tɔw fanba la (Ja S13), a ye Angstrom 3,8 ye ka bɔ Q1 ka oksizɛni metili la, wa a bɛ se ka hidrɔzɛni jɛɲɔgɔnya barikamaw di (Jaa 5E). A bɛ iko Q3 tɛ ni jɛɲɔgɔnya kɛrɛnkɛrɛnnen ye, nka a bɛ jibɔyɔrɔ la RC-M yɔrɔ fitinin ni LH1-α yɔrɔ 5 fo 6 cɛ (Jaa 5, D ani G). Q1, Q2 ani Q3 walima kininoni kelalenw minnu bɛ u kɛrɛfɛ, olu fana ɲɛnabɔra Tch. Dususuman, Trv. Sɛgɛsɛgɛli 970 ani Blc. Iris jɔcogo (9, 10, 12) bɛ kinon dɛmɛnan siriyɔrɔ maralen dɔ jira RC-LH1 complexe kɔnɔ (Ja S12G). UQ duuru tolilen minnu bɛ RC-LH116 kɔnɔ, olu bɛ bɛn kosɛbɛ ni 5,8±0,7 ye complexe kelen-kelen bɛɛ la min latigɛra ni high performance liquid chromatography (HPLC) ye, k’a sɔrɔ UQ tolilen saba minnu bɛ RC-LH114-W kɔnɔ, olu ka dɔgɔ ni The measured value of 6,2±0,3 (Ja S14) b’a jira ko UQ molecules ma ɲɛnabɔlenw bɛ structure kɔnɔ.
L ni M polipɛtidi pseudo-symétrique kelen-kelen bɛɛ kɔnɔ, TMH duuru bɛ ​​sɔrɔ u kelen-kelen bɛɛ la, wa u bɛ kɛ hɛtɛrodimɛri ye min bɛ BChl dimɛri kelen, BChl monomɛri fila, bacteriophage (BPh) monomɛri fila, ani nɛgɛ kelen min tɛ Heme ye ani UQ10 mɔlikulu kelen walima fila fara ɲɔgɔn kan. Hidrɔzɛni jɛɲɔgɔnyaw sɔrɔli fɛ ketɔn kulu laban kan ani a dalajɛ dɔnta Rps kɔnɔ, karotɛnoyiw bɛ don M-subunit kɔnɔ, o min tɔgɔ ye ko cis-3,4-dehydroorhodopin. A suguya (25). RC-H kɛnɛma-yɔrɔ bɛ siri membrane la ni TMH kelen ye. RC jɔcogo bɛɛ lajɛlen bɛ tali kɛ suguya RC fitinin saba la minnu bɛ tali kɛ ɲɔgɔn na (i n’a fɔ Rba). sɔgɔsɔgɔninjɛ (PDB ID: 3I4D). BChl ni BPh ka makɔrɔsikiliw, karotɛni kɔkolo ni nɛgɛ min tɛ hɛmɛ ye, olu bɛ ɲɔgɔn dafa nin jɔli ninnu ka ɲɛnabɔli hakɛ kɔnɔ, i n’a fɔ UQ10 kunkolo kulu bɛ kɛ cogo min na QA yɔrɔ la ani QB kinɔni bɛ kɛ cogo min na RC-LH116 la (Jaa S15).
RC jɔcogo fila sɔrɔli ni QB yɔrɔ minɛni hakɛ tɛ kelen ye, o bɛ sababu kura di walasa ka fɛn caman sɛgɛsɛgɛ cogoya caman yeli caman na minnu bɛ taa ni QB kinɔni sirili ye. RC-LH116 complexe kɔnɔ, QB kinone bɛ sɔrɔ “proximal” yɔrɔ la min sirilen don kosɛbɛ (26), nka RC-LH114-W farali tɛ QB kinone sɔrɔ. QB kinɔni tɛ RC-LH114-W kɔnɔ, o ye kabako ye bawo complexe bɛ baara kɛ, ka tɛmɛ RC-LH116 complexe kan ni structurally resolved QB kinone ye. Hali ni LH1 bololanɛgɛ fila bɛ kela kinɔn wɔɔrɔ ɲɔgɔn na, duuru bɛ ​​ɲɛnabɔ jɔcogo la RC-LH116 bololanɛgɛ datugulen kɔnɔ, k’a sɔrɔ saba dɔrɔn de dan ye jɔcogo la RC-LH114-W bololanɛgɛ dafalen kɔnɔ Nin sigicogo gɛlɛya min cayara, o bɛ se ka kɛ sababu ye ka RC-LH114-W QB yɔrɔw caman cili teliya, kinone kinetiki teliyalen complexe kɔnɔ, ani ka LH1 loop tigɛcogo caya. An b’a jira ko UQ dɛsɛ RC-LH114-W ka RC QB yɔrɔ la, o bɛ se ka kɛ fɛn ye min ka gɛlɛn kosɛbɛ ani min bɛ baara kɛ kosɛbɛ, wa RC-LH114-W ka QB yɔrɔ sumayalen don o yɔrɔnin bɛɛ UQ jiginni na Etape kɛrɛnkɛrɛnnen (QB yɔrɔ dondaw datugulen don) bɛ nin baara in cogoya jira.
Ni QB tɛ, L-Phe217 jiginni min bɛ taa a fɛ ka taa yɔrɔ la min ni UQ10 sirili tɛ bɛn, bawo o bɛna kɛ sababu ye ka yɔrɔko ɲɔgɔn gosi ni kɔkolo isoprene unit fɔlɔ ye (Jaa 6A). Ka fara o kan, fɛnɲɛnɛmafagalanw yeli kunbaba jɛlenw bɛ ye, kɛrɛnkɛrɛnnenya la helix de (helix surun min bɛ TMH D ni E cɛ loopu la) yɔrɔ min na L-Phe217 bɛ wuli ka taa QB siriyɔrɔ la ani L-Tyr223 jiginni (Ja 6A ) Walasa ka hidrɔzɛni jɛɲɔgɔnya tiɲɛ ni M-Asp45 karamɔgɔya ye ani ka QB siriyɔrɔ da datugu (Jaa 6B) la. Helix de pivots bɛ a da la, L-Ser209 ka Cα bɛ Yɛlɛma ni 0,33Å ye, ka sɔrɔ L-Val221Cα bɛ Yɛlɛma ni 3,52Å ye. Yɛlɛma kɔlɔsilen si tɛ TMH D ni E la, minnu bɛ se ka bila ɲɔgɔn kan o jɔcogo fila bɛɛ la (Jaa 6A). An b’a Dɔn cogo min na, nin ye jɔli fɔlɔ ye RC nafama na min bɛ QB yɔrɔ datugu. Ni an y’a suma ni dafalen (QB-bound) sigicogo ye, o b’a jira ko sani kinin ka dɔgɔya, a ka kan ka fɛn dɔ Changer conformational (Yɛlɛma) walasa a ka don kinone kɔnɔ. L-Phe217 bɛ wuli ka π-stacking jɛɲɔgɔnya kɛ ni kinone kunkolo kulu ye, ani helix bɛ wuli ka taa kɛnɛma, o b’a to L-Gly222 kolosinsinnan ni L-Tyr223 kɛrɛfɛ cakɛda bɛ se ka hidrɔzɛni jɛɲɔgɔnya rezo dilan ni hidrɔzɛni jɛɲɔgɔnya jɔcogo sabatilen ye (Jaa 6, A ani C).
(A) Ologramu (L cakɛda, orange/M cakɛda, magenta) ani apo (gris) jɔcogo dessin animé min bɛ ɲɔgɔn kan, o kɔnɔ, tolen kolomaw bɛ jira cogo la min bɛ i n’a fɔ bere. UQ10 bɛ jira ni bara jɛman ye. Layini nɔrɔlen bɛ hidrɔzɛni jɛɲɔgɔnyaw jira minnu bɛ sɔrɔ o jɔcogo bɛɛ la. (B ani C) Apolipoprotéine ni bololanɛgɛ bɛɛ jɔcogo sanfɛla jiracogo, min bɛ L-Phe217 kɛrɛfɛ cakɛda oksizɛni jira ni bulu ye ani L-Tyr223 ni bilen ye. L yɔrɔ fitinin ye oranjɛ ye; M ni H yɔrɔ fitininw tɛ kulɛri ye. .
A ma kɛ cogo min na, hali ni QB dɛsɛbagatɔ RCw jɔcogo caman bɛ sɔrɔ minnu tɛ LH1 ye, fɛn minnu kɛra cogoya caman yeli ye minnu kɔlɔsira nin kalan in kɔnɔ, olu ma fɔ fɔlɔ. Olu dɔw ye QB depletion structure ye ka bɔ Blc. viridis (PDB ID: 3PRC) (27), Ɲɛgɛnɛsiraw ani mɔgɔ dogoyɔrɔw. tepidum (PDB ID: 1EYS) (28) ani Rba. sphaeroides (PDB ID: 1OGV) (29), olu bɛɛ ni u ka QB jɔcogo bɛɛ lajɛlen bɛ se ka kɛ kelen ye. 3PRC sɛgɛsɛgɛli ɲuman y’a jira ko LDAO (Lauryl Dimethyl Amine Oxide) safinɛ mɔlikuluw bɛ siri QB jɔyɔrɔ da la, o bɛ se ka kɛ sababu ye ka u labɛn kokura ka kɛ cogoya dagalen ye. Hali ni LDAO tɛ toli yɔrɔ kelen na 1EYS walima 1OGV kɔnɔ, o RC ninnu bɛ labɛn ni safinɛ kelen ye, o la u bɛ se ka nɔ kelen bɔ. Rba ka kristalɛ jɔcogo. Sphaeroides RC co-crystallized ni cytochrome c2 (PDB ID: 1L9B) fana bɛ i n’a fɔ QB yɔrɔ datugulen bɛ u la. Nka, o cogo la, RC-M polipɛtidi ka N-terminal yɔrɔ (min bɛ jɛɲɔgɔnya kɛ ni QB siriyɔrɔ ye Tyr tolen ka H jɛɲɔgɔnya fɛ Q helix kan) bɛ cogoya dɔ ta min tɛ a yɛrɛ ta ye, wa QB cogoya caman yeli tɛ sɛgɛsɛgɛ ka taa a fɛ (30 ). Min bɛ mɔgɔ hakili sigi, o ye ko an ma nin M polipɛtidi bɔgɔlan sugu ye RC-LH114-W jɔcogo la, min ni RC-LH116 RC N-terminal yɔrɔ bɛ Taa ɲɔgɔn fɛ. A ka kan fana k’a dɔn ko LH1 antɛni min sinsinnen bɛ safinɛ kan, o bannen kɔfɛ, apolipoprotéine RC minnu bɛ PDB kɔnɔ, olu ɲɛnabɔra, o min ye kininɔn kɔnɔna jidagayɔrɔw ni tulumafɛnw bɔ RC ni LH1 bololanɛgɛ lamini kɔnɔna cɛ (31, 32). RC bɛ to baara la bawo a bɛ kofactor bɛɛ mara, fo QB kinone min bɛ se ka tiɲɛ, o tɛ sabati kosɛbɛ, wa a ka teli ka tunun a labɛnni senfɛ (33). Ka fara o kan, a dɔnna ko LH1 ni tulumafɛnw bɔli RC la, o bɛ se ka nɔ bila baarakɛcogo la, i n’a fɔ P+QB-cogo min bɛ faranfasi ni charge ye, o ka ɲɛnamaya surunya (31, 34, 35). O la, an b’a miiri ko sigida LH1 bololanɛgɛ min bɛ RC lamini, o sɔrɔli bɛ se ka QB yɔrɔ “dasirilen” mara, o la, sigida lamini bɛ lakana QB kɛrɛfɛ.
Hali ni apolipoprotéine (ni QB kinone tɛ) ni a jɔcogo dafalen bɛ QB yɔrɔ jiginni ja fila dɔrɔn de jira, sanni ka kɛ ko kɛlenw ye minnu bɛ tugu ɲɔgɔn na, taamasiɲɛw bɛ yen minnu b’a jira ko sirili bɛ se ka da walasa ka sirili bali kokura ni hidrokinɔni ye Walasa ka substrat balili bali. Kinɔlɔli ni kinɔni ka ɲɔgɔn sɔrɔ apolipoprotéine QB yɔrɔ kɛrɛfɛ, o bɛ se ka kɛ danfara ye, o bɛ na ni a ban ye RC fɛ. A fɔra kabini tuma jan ko fɛnɲɛnɛma caman yeli jɔyɔrɔ ka bon kinɔnw sirili n’u dɔgɔyali la. RC minnu sumana, olu ka se ka kinonw dɔgɔya dibi ladamuni kɔfɛ, o bɛ tiɲɛ (36); X-ray crystallography y’a jira ko nin tiɲɛni in sababu bɔra QB kinonw sirili la “distal” conformation kɔnɔ 4,5 Å ɲɔgɔn ka bɔ active proximal position (26) , 37). An b’a jira ko nin yɔrɔjan sirili cogoya in ye cɛmancɛ cogoya ja ye apolipoprotéine ni ring structure dafalen cɛ, o min bɛ tugu jɛɲɔgɔnya fɔlɔ la ni kinone ye ani QB yɔrɔ dabɔli.
RC suguya II min bɛ sɔrɔ PSII complexe kɔnɔ fototalan dɔw ni siyanobakteriw, jɛgɛw ani jiriw la, o bɛ ni jɔcogo ni baarakɛcogo lakanani ye (38). Jatebla 6 (D ni E) kɔnɔ, sigicogo sinsinni bɛ sinsin PSII RCw ni banakisɛw ka RC complexe QB yɔrɔ cɛ ɲɔgɔndɛmɛ kan. O sangaɲɔgɔnma in kɛra misali ye kabini tuma jan walasa ka kinɔn sirili ni a dɔgɔyali cogoyaw sɛgɛsɛgɛ minnu bɛ tali kɛ ɲɔgɔn na kosɛbɛ. Sɛbɛn tɛmɛnenw y’a jira ko fɛnɲɛnɛma caman yeli bɛ taa ni PSII dɔgɔyali ye kinɔnw na (39, 40). O la, n’i y’a jateminɛ ko RC lakanani jiginni siratigɛ la, nin siricogo in min tun ma kɔlɔsi fɔlɔ, o bɛ se ka kɛ fana PSII RC ka QB yɔrɔ la fototalan jiriw la minnu bɛ ni oksizɛni ye.
Rps ΔpufW (pufW bɔli min ma taamasiyɛn kɛ) ani PufW-His (C-terminal 10x His-tagged protein-W min jiralen don ka bɔ pufW yɔrɔ nafama na) suguyaw. palustris CGA009 ɲɛfɔra an ka baara tɛmɛnenw na ( 16 ). Nin suguya ninnu ni kungo kɔnɔ bangebaga isojɛni sɔrɔla ka bɔ konfirɛzi kɔnɔ ni selilu damadɔw kɛli ye PYE (u kelen-kelen bɛɛ ye garamu 5 litiri -1) (minnu maralen bɛ LB kɔnɔ -80 °C la, 50% ( w/v) glycérol) proteine, yeast extract ani succinate) agar [1,5% (w/v)] plati bɛ min na. Aw bɛ plati in bila su dibi la so funteni na ni fiɲɛ tɛ a la, o kɔfɛ, a yeelenna ni yeelen finman ye (~50 μmolm-2 s-1) min bɛ di OSRAM 116-W halogène anpuluw fɛ (RS Components, UK) tile 3 fo tile 5 fo ka se koloni kelen ka bɔ. Koloni kelen kɛra ka M22+ fɛnɲɛnamafagalan mililitiri 10 (41) pikiri kɛ min farala asidi kasamini 0,1% (kg/v) kan (a bɛ fɔ a ma kɔfɛ ko M22). Sɛnɛfɛn in falenna oksizɛni dɔgɔyalen na dibi la 34°C la ni a yɛrɛyɛrɛli ye ni 180 ye miniti kɔnɔ sanga 48 kɔnɔ, o kɔfɛ, sɛnɛfɛn mililitiri 70 pikirila o cogo kelen na fo lɛri 24. Semi-aerobic culture min hakɛ ye mililitiri 1 ye, o bɛ kɛ ka M22 fɛnɲɛnamafagalan mililitiri 30 pikiri mililitiri 30 universel screw-top transparent glass bottle kɔnɔ ka sɔrɔ ka fiɲɛ bila a la ni wulikan ye (~50μmolm-2 s-1) fo lɛri 48 ni magnɛti fanga sterili ye Stirring rod. O kɔfɛ, mililitiri 30 kɛra sɛnɛfɛn na ni sɛnɛfɛn litiri 1 ɲɔgɔn ye o cogo kelen na, o kɔfɛ, o kɛra ka sɛnɛfɛn litiri 9 ɲɔgɔn pikiri min yeelenna ~200 μmolm-2 s-1 la sanga 72 kɔnɔ. Bugunw lajɛra ni centrifugation ye 7132 RCF la miniti 30 kɔnɔ, ka segin ka u su ~10 ml 20 mM tris-HCl (pH 8.0) la, ka u mara -20°C la fo ka se u mago ma.
Aw bɛ a funu kɔfɛ, aw bɛ deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) ani Roche holoenzyme protease inhibiteur furakisɛ fila (Merck, UK) kristalɛ dɔw fara farikolokisɛw kan minnu bɛ segin ka wuli. Faransi tansiyɔn selilɛri 20 000 psi kɔnɔ (Aminco, Etazuni), seliluw tiɲɛna siɲɛ 8 fo siɲɛ 12. Selilu minnu ma kari ani nɔgɔ minnu tɛ se ka wuli, olu bɔlen kɔ ni centrifugation ye 18.500 RCF la miniti 15 kɔnɔ 4°C la, membrane in jalen don ka bɔ lysate pigmenté la ni centrifugation ye 113.000 RCF la sanga 2 kɔnɔ 43.000°C la. Aw bɛ a yɔrɔ min bɛ se ka wuli, aw bɛ o fili ka segin ka ɲɛgɛnɛsira kulɛrilen 20 mM tris-HCl (pH 8,0) mililitiri 100 fo 200 kɛ ka sɔrɔ ka a kɛ kelen ye fo ka se a fɛnɲɛnamafagalanw ma minnu bɛ ye. Aw bɛ fɛnɲɛnamafagalan jɔlen tobi tris-HCl 20 mM (pH 8.0) (Anatrace, Etazuni) la, β-DDM 2% (kg/v) bɛ min na fo lɛri 1 dibi la 4°C la ni a wulilen ye dɔɔni dɔɔni. O kɔ, aw bɛ centrifuge kɛ 70°C la walasa ka RCF 150.000 wuli 4°C la lɛri 1 kɔnɔ walasa ka tolenw bɔ minnu tɛ se ka wuli.
Solubilizante membrane min bɔra ΔpufW suguya la, o kɛra DEAE Sepharose ion exchange column mililitiri 50 la ni kolosinsinnan hakɛ saba (CV) ye [20 mM tris-HCl (pH 8,0) min kɔnɔ 0,03% (w / v) β-DDM bɛ sɔrɔ Aw bɛ kolotuguda ko ni CV sirilan fila ye, o kɔ aw bɛ kolotuguda ko ni sirilan fila ye minnu kɔnɔ NaCl 50 mM bɛ sɔrɔ. RC-LH116 complexe ko ni gradient lineaire ye min ye 150 fo 300 mM NaCl ye (binding buffer kɔnɔ) CV 1,75 kan, ka sirili complexe tɔ ko ni sirili tampon ye min kɔnɔ NaCl 300 mM bɛ sɔrɔ CV 0,5 kan. Aw bɛ absorption spectrum lajɛ 250 ni 1000 nm cɛ, ka fraction min absorbance ratio (A880/A280) ka ca ni 1 ye nm 880 fo 280 la, ka a dilan siɲɛ fila sirili tampon kɔnɔ, ka o kɛcogo kelen kɛ tuguni DEAE column kan On purification. Aw bɛ faranfasi minnu ka A880/A280 hakɛ ka ca ni 1,7 ye ani A880/A805 hakɛ ka ca ni 3,0 ye, aw bɛ ionw ɲɔgɔn falen-falen siɲɛ sabanan kɛ, ka tilayɔrɔ minnu ka A880/A280 hakɛ ka ca ni 2,2 ye ani A880/A805 hakɛ ka ca ni 5,0 ye, aw bɛ olu mara. Komplekisi min saniyalen don yɔrɔ dɔ la, o kɛra ~2 ml ye Amicon 100.000 molecular weight cut-off (MWCO) centrifugal filter (Merck, UK) kɔnɔ, ka sɔrɔ ka doni Superdex 200 16/600 size exclusion column (GE Healthcare, US) kan, NaCl tampon 200 mM bɛ min kɔnɔ, ka sɔrɔ ka a ko o tampon kelen na ni 1,5 CV ye. Aw bɛ hakɛ bɔli tilayɔrɔba ka minɛni spekterw lajɛ, ka absorption spectres lajɛ ni A880/A280 hakɛw ye 2,4 kan ani A880/A805 hakɛw bɛ 5,8 kan ka se 100 A880 ma, ka sɔrɔ ka baara kɛ n’u ye o yɔrɔnin bɛɛ ka cryo-TEM grid labɛnni walima a marali kɛ Aw bɛ a mara -80°C la fo ka se a mago ma.
PufW-His suguya min bɛ wuli, o kɛra HisPrep FF Ni-NTA Sepharose koloma mililitiri 20 na (20 mM tris-HCl (pH 8,0) min kɔnɔ NaCl 200 mM ani 0,03% (kg/k) bɛ sɔrɔ IMAC tampon kɔnɔ ( GE Healthcare). v) β-DDM) ye. Kolon ko ni IMAC tampon CV duuru ye, o kɔfɛ ni IMAC tampon CV duuru ye minnu kɔnɔ histidine 10 mM bɛ sɔrɔ. Core complexe (kɔnɔna gɛlɛn) bɔra kolosinsinnan na ni IMAC tampon duuru ye minnu kɔnɔ histidine 100 mM bɛ sɔrɔ. RC-LH114-W complexe bɛ yɔrɔ min na, o bɛ kɛ ka ~10 ml kɛ jidagayɔrɔ la min bɛ wuli ni Amicon 100.000 MWCO filɛri ye (Merck, UK), ka a wuli siɲɛ 20 ni sirili tampon ye, ka sɔrɔ ka fara mililitiri 25 kan DEAE Sepharose koloma kɔnɔ, CV naani sirilen bɛ tampon na, olu bɛ kɔn ka kɛ. Aw bɛ kolotuguda ko ni CV sirilan naani ye, o kɔ aw bɛ complexe in ko CV 8 kan ni gradient lineaire ye min ye 0 fo 100 mM NaCl ye (binding buffer kɔnɔ), ani CV naani tɔw minnu bɛ 100 mM sirili tampon na Complex tolenw kolilen don chlorure sodium kan faralen ɲɔgɔn kan ni A880/A280 hakɛ ye min ka ca ni 2,4 ye ani a A880/A805 hakɛ min ka ca ni tilayɔrɔ 4,6 ye, o kɛra ~2 ml ye Amicon 100.000 MWCO centrifuge filɛri kɔnɔ, ka sɔrɔ ka fa ni 1,5 CV IMAC ye ka kɔn Buffer equilibrated Superdex 200 16/600 size exclusion column kɔnɔ, ka sɔrɔ ka u ko tampon kelen na 1,5 CV kan. Aw bɛ hakɛ-bɔli yɔrɔw ka minɛni spekterw lajɛ ani ka absorption spectres lajɛ ni A880/A280 hakɛw ye 2,1 kan ani A880/A805 hakɛw bɛ 4,6 kan ka se 100 A880 ma, minnu bɛ kɛ o yɔrɔnin bɛɛ ka TEM grid congelé labɛn walima ka mara -80°C la fo mago bɛ u la.
Leica EM GP jisumalan dɔ kɛra ka TEM gridw labɛn minnu funteni ka dɔgɔn. O kɔmplɛsi in kɛra IMAC tampon na ka se A880 ma min ye 50 ye, o kɔfɛ, 5μl bilala QUANTIFOIL 1.2/1.3 kuran sɔgɔlen kura la min bɛ yeelen bɔ (Agar Scientific, UK). Aw bɛ gridi in bila 20°C la ani 60% nɛnɛ na fo s 30, o kɔ aw bɛ a ko ka ja fo s 3, ka sɔrɔ k’a faga etan jilama la -176°C la.
RC-LH114-W complexe ka kunnafoniw sɛbɛnna eBIC (Electronic Bioimaging Center) (British Diamond Light Source) kan ni Titan Krios mikroskɔpu ye, min bɛ baara kɛ ni teliya ye min ye 300kV ye, ni a bonya tɔgɔlama ye 130.000× ye ani a fanga ye- Choose a gap of 20 eV. Gatan 968 GIF Quantum ni K2 peak detector kɛra ka jaw sɛbɛn jatebɔcogo la walasa ka kunnafoniw lajɛ. Pixel hakɛ jateminɛlen ye 1,048Å ye, ani a tata hakɛ ye 3,83 e-Å-2s-1 ye. A ye filimu in lajɛ segin 11 kɔnɔ k’a tila tilayɔrɔ 40 ye. Aw bɛ baara kɛ ni yɔrɔ ye min datugulen don ni karɔbɔli ye walasa ka mikroskɔpu sinsin kokura, o kɔ aw bɛ filimu saba lajɛ dingɛ kelen kɔnɔ. K’a bɛɛ lajɛlen ta, filimu 3130 de lajɛra, ni defocus hakɛw bɛ -1 ni -3μm cɛ.
RC-LH116 complexe kunnafoniw lajɛra ni o mikroskɔpu kelen ye Asterbury Biostructure Laboratory (University of Leeds, UK) la. Kunnafoni ninnu lajɛra jatebɔ cogo la ni bonya ye 130 k ye, ani pixel hakɛ jateminɛna ka se 1,065 Å ma ni a tata hakɛ ye 4,6 e-Å-2s-1 ye. O filimu minɛna segin 12 kɔnɔ, ka tila tilayɔrɔ 48 ye. K’a bɛɛ lajɛlen ta, filimu 3359 de lajɛra, ni defocus hakɛw bɛ -1 ni -3μm cɛ.
Donanw baara bɛɛ bɛ kɛ Relion 3.0 pibiliki kɔnɔ (42). Aw bɛ Motioncorr 2 (43) kɛ ka biɲɛ lamagacogo latilen ni a tata hakɛ girinya ye, o kɔ aw bɛ CTFFIND 4.1 (44) kɛ ka CTF (contrast transfer function) paramɛtiri dɔn. Fotomikɔrɔgrafu minnu bɛ kɛ cogoya la nin baarakɛcogo fɔlɔ ninnu kɔfɛ, olu jiralen bɛ ja 2 na. S16. Otomatiki sugandili misali bɛ dilan ni bolo ye ka fɛnɲɛnamafagalan 1000 pixel 250 ɲɔgɔn sugandi pixel 250 frame kɔnɔ ani reference fila-fila (2D) danfara tɛ, o la, a bɛ ban o danfara minnu bɛ bɛn sample nɔgɔ ma walima minnu tɛ ni jogo ye min bɛ se ka dɔn. O kɔfɛ, otomatiki sugandili kɛra mikrofoto bɛɛ kan, ani RC-LH114-W tun ye fɛnɲɛnamafagalan 849 359 ye, ani RC-LH116 complexe tun ye fɛnɲɛnamafagalan 476 547 ye. Fɛnɲɛnɛma sugandilenw bɛɛ ye 2D danfara siɲɛ fila kɛ, wa boli kelen-kelen bɛɛ kɔfɛ, fɛnɲɛnamafagalan minnu bɛ bɛn karɔbɔli yɔrɔ ma, misali nɔgɔli, fɛn jɛlenw tɛ minnu na walima fɛnɲɛnɛma minnu bɛ ɲɔgɔn kan kosɛbɛ, olu bɛ ban, o bɛ na ni 772.033 (90,9%) ani 359.678 (75,5%) ye ) Fɛnɲɛnɛmaw bɛ kɛ ka 3D danfara kɛ RC-LH114-W ani RC-LH116 ye ɲɔgɔn sɔrɔ. 3D reference model fɔlɔ dabɔra ni stochastic gradient descent fɛɛrɛ ye. Ni an bɛ baara kɛ ni modɛli fɔlɔ ye i n’a fɔ kunnafoni, fɛnɲɛnɛma sugandilenw bɛ tila ka kɛ kulu naani ye 3D kɔnɔ. Ni aw bɛ baara kɛ ni modɛli ye min bɛ nin kulu in kɔnɔ i n’a fɔ kunnafoni, ka 3D sɛgɛsɛgɛli kɛ fɛnɲɛnamafagalanw kan minnu bɛ kuluba kɔnɔ, o kɔfɛ, aw bɛ baara kɛ ni 15Å low-pass filɛri fɔlɔ ye walasa ka fɛnɲɛnamafagalan yɔrɔ datugu, ka pixel 6 fara a da nɔgɔlenw kan, ka pixelw baara kɔfɛ walasa ka Gatan K2 peak Modulation transfer function latilen sanfɛ detektɛri la. RC-LH114-W kunnafonidilan ta fan fɛ, nin misali fɔlɔ in sɛmɛntiyalen don ni density barikama bɔli ye masike dakunw na (a tigɛlen don ni core complex density ye UCSF Chimera kɔnɔ). O misali minnu sɔrɔla (RC-LH114-W ni RC-LH116 ka ɲɛfɔliw ye 3,91 ani 4,16 Å ye), olu bɛ kɛ fɛn ye min bɛ kɛ ka ɲɛsin 3D danfara filanan ma. Fɛnɲɛnɛma minnu bɛ Kɛ, olu bɛ Jɛ ka Kɛ 3D kalasi fɔlɔ ye, wa jɛɲɔgɔnya barikama tɛ u ni sigida la. Daɲɛw sigicogo jɛlenw bɛ ɲɔgɔn dafa walima u dɛsɛ. 3D danfara filanan kɔfɛ, kulu min ka ɲɛfɔli ka bon kosɛbɛ, o de sugandira [RC-LH114-W ta la, kulu kelen ye fɛnɲɛnamafagalan 377.703 ye (44,5%), RC-LH116 ta la, kulu fila bɛ yen, u bɛɛ lajɛlen ye fɛnɲɛnamafagalan 260.752 ye (54,7%) , Yɔrɔ min na u bɛ kɛ kelen ye dɔrɔn n’u bɛ bɛn ɲɔgɔn ma jiginni fɔlɔ kɔfɛ ni danfara fitinin ye Fɛnɲɛnɛma sugandilenw bɛ segin ka bɔ kɛsu kɔnɔ min janya ye 400 ye, ka u nɔgɔya ni 3D sɛgɛsɛgɛli ye. Solvent mask bɛ dilan ni 15Å low-pass filɛri fɔlɔ ye, 3 pixel karti expansion ani 3 pixel soft mask. Ni baara kɛra ni CTF sɛnɛfɛn kelen-kelen bɛɛ labɛnni ye, fɛnɲɛnamafagalan kelen-kelen bɛɛ lamagacogo latilenni ani fɛnɲɛnamafagalan kelen-kelen bɛɛ labɛnni CTF sɛnɛcogo filanan, 3D labɛnni, solvent masking ani post-processing bɛ kɛ sen kelen-kelen bɛɛ kɔfɛ walasa ka fɛn dilannenw nɔgɔya ka taa a fɛ. Ni baara kɛra ni FSC (Fourier Shell Correlation Coefficient) tigɛda hakɛ ye min ye 0,143 ye, RC-LH114-W ni RC-LH116 ka misali labanw ka ɲɛfɔliw ye 2,65 ani 2,80Å ye. FSC kurufe min bɛ modɛli laban na, o jiralen bɛ ja 2 na. S17.
Protéines sinsinni bɛɛ bɛ telesarse ka bɔ UniProtKB la: LH1-β (PufB; UniProt ID: Q6N9L5); LH1-α (PufA; UniProtID: Q6N9L4) ye baara kɛ ni a ye; RC-L (PufL; UniProt ID: O83005); RC-M (PufM; UniProt ID: A0A4Z7) ye baara kɛ ni a ye; RC-H (PuhA; UniProt ID: A0A4Z9); Protéine-W (PufW; UniProt ID: Q6N1K3) ye fɛn ye min bɛ se ka kɛ. SWISS-MODEL (45) kɛra ka RC ka homologie modɛli dɔ dilan, min kɔnɔ RC-L, RC-M ani RC-H ka poroteyini sinsinniw bɛ sɔrɔ ani Rba ka kristalɛ jɔcogo. sphaeroides RC kɛra misali ye (PDB ID: 5LSE) (46). Aw bɛ baara kɛ ni “fit map” baarakɛminɛn ye UCSF Chimera kɔnɔ walasa ka modeli dilannen bɛn karti (47) ma, ka farikolojɔlifɛnw sigicogo ɲɛ, ani ka cofactor [4×BChl a (monomer library residue name = BCL), 2×BPh a (BPH), UQ10 suguya kelen walima fila (U10), nɛgɛ kelen min tɛ hɛmɛ ye (Fe) ani 3,4-dihydrohexacarbonylcholine kelen (QAK)] bɛ Coot (48) kɛ ka fara a kan. Ikomi QAK tɛ sɔrɔ monomɛri gafemarayɔrɔ la, a kɛra paramɛtiri ye ni eLBOW baarakɛminɛn ye PHENIX kɔnɔ (49).
O kɔfɛ, LH1 yɔrɔ fitinin in jɔra. A daminɛ na, otomatiki jɔli baarakɛminɛn min bɛ PHENIX (49) kɔnɔ, o kɛra sababu ye ka LH1 sinsinni yɔrɔ dɔ jɔ a yɛrɛma ni karti ye ani LH1-α ni LH1-β poroteyini sinsinniw i n’a fɔ donta. LH1 yɔrɔ fitinin min dafalen don kosɛbɛ, o sugandi, k’a bɔ k’a doni Coot kɔnɔ, ka ɲɔgɔndan tununnen fara a kan ni bolo ye, ka bololabaara kɛ ka a jɔcogo bɛɛ labɛn ka sɔrɔ ka BCl fila a (BCL) ni spirilloxanthin (CRT) fara a kan [ka kɛɲɛ ni Rps ye min bɛ tali kɛ o la LH1 complexe density ani carotenoïde hakɛ dɔnta. A suguya (17)]. LH1 yɔrɔ fitinin dafalen kopi, ka baara kɛ ni UCSF Chimera “Docking Map Tool” ye walasa ka dock kɛ LH1 density yɔrɔ la min tɛ modeli ye, o kɛrɛfɛ, ka sɔrɔ k’a labɛn Coot kɔnɔ aw bɛ segin o kɛcogo kan fo LH1 yɔrɔ fitininw bɛɛ ka kɛ misali ye. RC-LH114-W sigicogo kama, ni density tilabali bɔ Coot kɔnɔ, poroteyini bɛ tila ka bɔ fɛn tɔw la minnu tɛ poroteyini ye USCF Chimera karti kɔnɔ ani Autobuild baarakɛminɛn bɛ kɛ ka misali fɔlɔ sigi sen kan, ani yɔrɔ fitinin tɔw (poroteyini-W) Modeling. FENIX (49) kɔnɔ. Aw bɛ ɲɔgɔndan minnu tununna, olu fara modeli sɔrɔlen kan Coot (48) kɔnɔ, o kɔfɛ, aw bɛ bololabaara kɛ ka yɔrɔ fitinin bɛɛ labɛn. Density tɔ min ma tila, o bɛ bɛn lipidiw (PDB monomère bibliothèque ID de CDL = CDL, POPC = 6PL et POPG = PGT), β-DDM detergent (LMT) ani UQ10 molecules (U10) faralen ɲɔgɔn kan. Baara kɛ ni PHENIX optimisation (49) ani manual optimisation ye Coot (48) kɔnɔ walasa ka modɛli fɔlɔ dafalen dafalen kɛ fo modeli jateminɛw ni fitinin yecogo ɲuman tɛ se ka ɲɛ ka taa a fɛ. A laban na, baara kɛ ni LocScale (50) ye walasa ka sigida karti nɔgɔya, o kɔfɛ, ka sɛgɛsɛgɛli damadɔ wɛrɛw kɛ ka density unalocated modeling ani automatic and manual optimization kɛ.
Pepitiriw, kofaktɛriw ani lipidi ni kinɔn wɛrɛw minnu bɛ jɛgɛn u ka density kɔnɔ, olu jiralen bɛ ja 1 ni 2 kɔnɔ.S18 fo S23. Modeli laban in jatebɔ kunnafoniw jiralen don jatebɔsen S1 kɔnɔ.
Fo n’a fɔra cogo wɛrɛ la, UV/Vis/NIR absorption spectres (UV/Vis/NIR absorption spectres) lajɛra Cary60 spectrophotometer (Agilent, Etazuni) kan, n’o ye 1 nm ye, k’a ta 250 nm na ka se 1000 nm ma ani 0,1s integration waati.
Aw bɛ sɛgɛsɛgɛli kɛ kuwari kubɛri kɔnɔ ni milimɛtɛrɛ 2 sira ye ka se A880 ma min ye 1 ye, ka sɔrɔ ka a minɛcogo spektrum lajɛ 400 ni 1000 nm cɛ. Sɛrɛkili dichroïque spectres circulares lajɛra Jasco 810 spectropolarimètre (Jasco, Japon) kan 1 nm cɛ 400 nm ni 950 nm cɛ ni scan rate ye 20 nm min-1.
Molar extinction coefficient bɛ dɔn ni core complexe dilution ye ka se A880 ma min bɛ se 50. Aw bɛ 10μl hakɛ dilan 990μl binding buffer walima methanol kɔnɔ, ka sɔrɔ ka absorption spectrum lajɛ o yɔrɔnin bɛɛ walasa ka BChl tiɲɛni dɔgɔya. BChl hakɛ min bɛ mɛtanɔli sɛgɛsɛgɛli kelen-kelen bɛɛ la, o jate kɛra ni tununi hakɛ ye min bɛ 771 nm na, o ye 54,8 mM-1 cm-1 ye, ani tununi hakɛ jateminɛna (51). Aw bɛ BChl hakɛ sumanlen tila ni 32 (RC-LH114-W) walima 36 (RC-LH116) ye walasa ka core complex hakɛ dɔn, o kɔfɛ, o bɛ kɛ ka o sɛgɛsɛgɛli kɛcogo kelen in minɛcogo dɔn min lajɛra tampon Extinction coefficient kɔnɔ. kuntilenna kelen. Sumani saba segin-segin kɛra sɛgɛsɛgɛli kelen-kelen bɛɛ la, wa BChl Qy maksimali absorbance moyenne kɛra jatebɔ kama. RC-LH114-W ka tununi hakɛ min suman ni 878 nm ye, o ye 3280±140 mM-1 cm-1 ye, ka sɔrɔ RC-LH116 ka tununi hakɛ min suman ni 880 nm ye, o ye 3800±30 mM-1 cm-1 ye.
UQ10 hakɛ jateminɛna ka kɛɲɛ ni fɛɛrɛ ye min bɛ (52) kɔnɔ. Kuma surun na, HPLC fasa kɔsegin (RP-HPLC) kɛra ni Agilent 1200 HPLC sitɛmu ye. Aw bɛ RC-LH116 walima RC-LH114-W 0,02 nmol ɲɔgɔn kɛ mɛtanɔli:kulorofɔrimu 50:50 50μl la, kuloridi feriki 0,02% (k/v) bɛ min na, ka sɔrɔ ka Beckman Coulter Ultrasphere ODS 4,6 mm min kɛra ka kɔn ka balanna, o pikiri kɛ mililitiri 1-1 min-1 la, 40°C la HPLC kɔnɔ furaji (80:20 mɛtanɔli:2-poropanɔli) bɛ kɛ ×25 cm koloma kan. Aw bɛ isocratic elution kɛ HPLC solvent kɔnɔ walasa ka absorbance kɔlɔsi 275 nm (UQ10), 450 nm (caroténoïdes) ani 780 nm (BChl) la sanga 1 kɔnɔ. A kuncɛyɔrɔ min bɛ 275 nm chromatogramme kɔnɔ miniti 25,5 kɔnɔ, o farala ɲɔgɔn kan, o min kɔnɔ, fɛn wɛrɛ minnu bɛ se ka dɔn, olu tun tɛ o la. Yɔrɔ jɛlen bɛ kɛ ka UQ10 bɔlen hakɛ molari jate ka kɛɲɛ ni jateminɛ kɛcogo ye min jate kɛra ka bɔ sariya saniyalenw pikiri la ka bɔ 0 na ka se 5,8 nmol ma (Jaa S14). Sɛgɛsɛgɛli kelen-kelen bɛɛ kɛra siɲɛ saba ye, wa fili min fɔra, o bɛ bɛn SD ma min bɛ sɔrɔ cɛmancɛ la.
Fura min kɔnɔ RC-LH1 complexe bɛ sɔrɔ ni Qy minɛcogo hakɛ cayalenba ye 0,1 ye, o labɛnna ni sow dusukun sitokɔrɔmu c2 dɔgɔyalen 30 μM ye (Merck, UK) ani 0 fo 50 μMUQ2 (Merck, UK). Militiri 1 sɛgɛsɛgɛli saba labɛnna UQ2 hakɛ kelen-kelen bɛɛ la, ka u bila su fɛ dibi la 4°C la walasa ka a jira ko u bɛ se ka ladamu pewu dibi la sani u ka sumani kɛ. O furaji donna OLIS RSM1000 modulari spektrofotomɛtiri kɔnɔ min kɔnɔna na, tasuma ye 300 nm/500 layini grating ye, 1,24 mm dondaw, 0,12 mm cɛmancɛ ani 0,6 mm bɔyɔrɔ sɔgɔlanw. Filɛri min janya ye 600 nm ye, o bɛ bila fototalan misali ni fototalan min bɛ tali kɛ a la, o da la walasa ka wuli yeelen bɔ. Absorbance kɔlɔsira 550 nm la ni integration waati ye 0,15 s. Lawulili yeelen bɛ bɔ 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) la ka tɛmɛ fibre optique kabɔri fɛ ni fanga ye 90% ye DC2200 controller fɛ (Thorlabs Ltd., UK) ani ka bɔ yeelenbɔyɔrɔ la ni 90° ye. Sumanikɛlan bɛ kɛlɛ ni filɛlikɛlan ye walasa ka yeelen fɛn o fɛn segin, yeelen fɛn o fɛn tun ma minɛ a daminɛ na misali fɛ. Aw bɛ a minɛcogo kɔlɔsi s 10 sani yeelen ka se s 50 ma. O kɔfɛ, a minɛni kɔlɔsira ka taa a fɛ fo s 60 dibi la walasa ka a dɔn kinololi bɛ sitokɔrɔmu c23 + dɔgɔya a yɛrɛ ma fo ka se hakɛ min ma (aw ye ja S8 lajɛ walasa ka kunnafoni raw sɔrɔ).
Kunnafoni ninnu kɛra ni daminɛ hakɛ sinsinni ye min bɛ bɛn s 0,5 fo 10 ma (ka kɛɲɛ ni UQ2 hakɛ ye) ani ka misali saba bɛɛ hakɛw cɛmancɛ kɛ UQ2 hakɛ kelen-kelen bɛɛ la. RC-LH1 hakɛ min jatera ni tununi hakɛ kelen-kelen bɛɛ ye, o kɛra sababu ye ka hakɛ caman sɛmɛntiya ka kɛ katalizi nafama ye, min jatera Origin Pro 2019 (OriginLab, Etazuni) kɔnɔ, ka bɛn Michaelis-Menten modɛli ma walasa ka Km ni Kcat hakɛ jiralenw dɔn.
Walasa ka waati kunkurunnin sumani kɛ, RC-LH1 sɛgɛsɛgɛli kɛra ka ~2μM kɛ IMAC tampon kɔnɔ, ascorbate sodium 50 mM (Merck, Etazuni) ani Terbutin 0,4 mM (Merck, Etazuni) bɛ min kɔnɔ. Asidi askorbiki bɛ kɛ ɛlektrɔniki sarakalan ye, ani tert-butaclofen bɛ kɛ QB balilan ye walasa ka a jira ko RC dilanbaga kunba ka to ka dɔgɔya (o kɔrɔ ye ko a tɛ fotooksidan sɔrɔ) sumani kɛcogo bɛɛ kɔnɔ. Militiri 3 ɲɔgɔn bɛ fara selilabana kan min bɛ wuli ka kɛɲɛ ni ladamu ye (a cɛmancɛ bɛ se mɛtɛrɛ 0,1 ma, 350 RPM) ni ɲɛnayeli sira janya ye milimɛtɛrɛ 2 ye walasa ka a jira ko sɛgɛsɛgɛlikɛlan min bɛ laser sira la, o ka waati bɛrɛ sɔrɔ dibi ladamuni na wulikanw ni ɲɔgɔn cɛ. Aw bɛ ~100-fs laser pulses kɛ ka Ti: Sapphire laser system (Spectra Physics, USA) fanga bonya walasa ka sample lawuli 880 nm la ni seginkanni hakɛ ye 1 kHz (20 nJ NIR ta la walima 100 nJ Vis ta la). Sani aw ka kunnafoniw lajɛ, aw bɛ sɛgɛsɛgɛlikɛlan bila nisɔndiya yeelen na miniti 30 ɲɔgɔn. A tali bɛna kɛ sababu ye ka QA baarakɛbaliya (a bɛ se ka kɛ ka QA dɔgɔya siɲɛ kelen walima siɲɛ fila). Nka aw k’a kɔlɔsi ko nin wale in bɛ se ka segin kɔ bawo dibi ladamuni waati jan kɔfɛ, RC bɛ segin dɔɔnin dɔɔnin QA baara la. Helios spectrometer (Ultrafast Systems, Etazuni) kɛra ka spectro transitoire suman ni latɛmɛ waati ye -10 fo 7000 ps . Baara kɛ ni Surface Xplorer porogaramu ye (Ultrafast Systems, Etazuni) walasa ka kunnafonidilanw fara ɲɔgɔn kan, ka sɔrɔ ka u fara ɲɔgɔn kan ani ka u kɛ sariya ye. Baara kɛ ni CarpetView porogaramu ye (Light Conversion Ltd., Lithuania) walasa ka baara kɛ ni kunnafonidilanw faralen ye ɲɔgɔn kan walasa ka danfaralanw sɔrɔ minnu bɛ tali kɛ toli la, walima ka baara kɛ ni baarakɛcogo ye min bɛ jiralan caman wuli ni minɛn jaabi ye walasa ka bɛn spektral jiginni kelen-kelen bɛɛ ma Origin kɔnɔ (OriginLab, Etazuni).
I n’a fɔ a fɔra cogo min na sanfɛ (53), fotosintetiki filimu dɔ labɛnna, LH1 complexe bɛ min kɔnɔ, RC ni LH2 antenne periférique fila bɛɛ tɛ min na. Aw bɛ membrane in dilan 20 mM tris (pH 8,0) la, ka sɔrɔ ka doni kɛ quartz cuvette kɔnɔ ni 2 mm ɲɛnayeli sira ye. Laser pulse 30nJ kɛra ka sɛgɛsɛgɛli kɛ ni 540 nm ye ni latɛmɛ waati ye -10 fo 7000 ps. Aw bɛ kunnafonidilanw baara i n’a fɔ a ɲɛfɔlen don cogo min na Rps. pal misali.
Membrane in kɛra bɔgɔlan ye ni centrifugation ye 150.000 RCF la sanga 2 kɔnɔ 4°C la, o kɔfɛ a absorbance 880 nm la, a bɛ segin ka suspension kɛ 20 mM tris-HCl (pH 8.0) ani 200 mM NaCl la. Aw bɛ membrane (minɛn) wuli ni aw bɛ a wuli dɔɔni dɔɔni β-DDM 2% (w/v) la lɛri 1 kɔnɔ dibi la 4°C la. Aw bɛ a lajɛ ni triethylammonium carbonate (pH 8,0) mililitiri 100 ye (TEAB; Merck, UK) fo ka se farikolojɔli dumuni hakɛ miligaramu 2,5 ma mililitiri-1 (Bio-Rad sɛgɛsɛgɛli). Baara wɛrɛw kɛra ka bɔ fɛɛrɛ la min bɔra ka tɛmɛ (54), k’a daminɛ ni farikolojɔli dumuni 50 μg dilan ye ka kɛ TEAB 50 μl bɛɛ lajɛlen ye, laurate sodium 1% (kg/v) bɛ min na (Merck, UK). Sonication kɔfɛ s 60 kɔnɔ, a dɔgɔyara ni 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) ye 37°C la miniti 30 kɔnɔ. Walasa ka S-alkylation sɔrɔ, aw bɛ a lajɛ ni 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) ye ka sɔrɔ ka a fara a kan ka bɔ 200 mM isopropanol stock solution la miniti 10 kɔnɔ so funteni na. Protéolytique digestion kɛra ni tripsin/endoprotéinase Lys-C ɲagaminen (Promega UK) miligaramu 2 farali ye a kan ka sɔrɔ ka a bila 37°C la sanga 3 kɔnɔ. Laurate surfactant bɔra ni etili asetati 50 μl ni 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) farali ye a kan ka sɔrɔ ka vortex kɛ s 60 kɔnɔ. Fasa farali yiriwara ni santirifugare ye 15.700 RCF la miniti 5 kɔnɔ. Ka kɛɲɛ ni a dilabaga ka sariya ye, C18 spin column (Thermo Fisher Scientific, UK) kɛra ka pepitiri bɛ duguma fasa min na, o sama ka ɲɛ ani ka kɔgɔ bɔ a la. Jalen kɔfɛ ni centrifugation vacuum ye, o sɛgɛsɛgɛli kɛra TFA 0,5% ni asetonitirili 3% la, ka ng 500 sɛgɛsɛgɛ ni nanoflow RP chromatography ye ka fara mass spectrometry kan ni sistɛmu paramɛtiriw ye minnu ɲɛfɔra ka tɛmɛ.
Aw bɛ MaxQuant v.1.5.3.30 (56) kɛ ka farikolojɔli dumuniw dɔn ani ka u hakɛ jateminɛ walasa ka Rps ɲini. palustris protéome kunnafonidilan (www.uniprot.org/proteomes/UP000001426). Mas spectrometry proteomics kunnafoniw bilala ProteomeXchange Alliance kɔnɔ PRIDE jɛɲɔgɔnw ka marayɔrɔ fɛ (http://proteomecentral.proteomexchange.org) kunnafonidilanw taamasiyɛn PXD020402 jukɔrɔ.
Walasa ka sɛgɛsɛgɛli kɛ ni RPLC ye ka fara electrospray ionization mass spectrometry kan, RC-LH1 complexe labɛnna ka bɔ kungo kɔnɔ Rps la. Ni an bɛ baara kɛ ni fɛɛrɛ ye min bɔra ka tɛmɛ (16), farikolojɔli dumuni hakɛ min bɛ sɔrɔ palustris selilɛriw kɔnɔ, o ye miligaramu 2 mililitiri-1 ye Hepes mililitiri 20 (pH 7,8), NaCl mililitiri 100 ani β- 0,03% (kg/v) (Bio-Rad sɛgɛsɛgɛli) ) DDM la. Ka kɛɲɛ ni a dilannikɛla ka sariya ye, aw bɛ saniyalan 2D (GE Healthcare, Etazuni) kɛ ka farikolojɔli dumuni 10 μg bɔ sanji kɛcogo la, ka jalen kɛ asidi formiki (FA) 20 μl 60% (v / v), asetonitirili 20% (v / v) ani ji 20% (v/v) la. Mikorolitiri duuru sɛgɛsɛgɛra ni RPLC (Dionex RSLC) ye ka fara mass spectrometry (Maxis UHR-TOF, Bruker) kan. Aw bɛ MabPac milimɛtiri 1,2×100 koloma (Thermo Fisher Scientific, UK) kɛ ka faranfasi 60°C ni 100μlmin -1 la, ni a jiginni ye 85% (v / v) furaji A [0,1% (v / v) FA ani 0,02% (V/v) TFA jilama] ka se 85%(v/v) furaji B ma [0,1%(v/v) FA ani 0,02%(v/v) asetonitirili TFA 90%(v/v) la] Ni aw bɛ baara kɛ ni ionizasiyɔn sɔrɔyɔrɔ ye min bɛ wele ko standard electrospray ionization source ani default parameters ka tɛmɛ miniti 60 kan, mass spectrometer bɛ 100 fo 2750 m/z sɔrɔ (mass-to-charge ratio). ExPASy biyoinformatiki nafolomafɛnw dakun FindPept baarakɛminɛn (https://web.expasy.org/findpept/) dɛmɛni fɛ, ka jamanadenw ka spektrum kariti kɛ ka taa complexe ka yɔrɔ fitininw na.
Bugunw falenna sanga 72 kɔnɔ NF-low (10μMm-2 s-1), mililitiri 100 yeelen na (30μMm-2 s-1) walima yeelenba (300μMm-2 s-1). M22 fɛnɲɛnamafagalan (M22 fɛnɲɛnamafagalan min na, amoniyɔmu sulfate bɛ bɔ a la ka sodium succinate bila a nɔ na ni sodiyɔmu asetati ye) mililitiri 100 buteli kɔnɔ min bɛ kɛ ni kurukuru ye (23). S 30-s cycle duuru kɔnɔ, 0,1 micron gilasi biɲɛw kɛra biɲɛ ye ni u hakɛ ye 1:1 ye walasa ka seliluw wuli ka u nɛnɛ jisuma kan miniti 5 kɔnɔ. Fɛn min tɛ se ka wuli, selilu minnu ma kari ani galasi biɲɛw bɔra ni centrifugation ye 16.000 RCF la miniti 10 kɔnɔ benchtop microcentrifuge kɔnɔ. Aw bɛ membrane fara ɲɔgɔn kan Ti 70,1 rotor kɔnɔ ni RCF 100.000 ye 20 mM tris-HCl (pH 8,0) la ni 40/15% (w/w) ye sucrose gradient ye sanga 10 kɔnɔ.
I n’a fɔ a ɲɛfɔra cogo min na an ka baara tɛmɛnenw na, immunodetection of the His tag on PufW (16). Kuma surun na, RC hakɛ kelen bɛ min kɔnɔ (11,8 nM) walima membrane min kɔnɔ RC hakɛ kelen bɛ sɔrɔ (o bɛ dɔn ni oksidan ye ka danfara spektrum dɔgɔyalen bɔ ani ka doni bɛn jeli nɔgɔlen kan) 2x SDS doni tampon kɔnɔ (Merck, UK) min bɛ dilan siɲɛ fila. Protéines (banakisɛfagalanw) farala ɲɔgɔn kan 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK) ɲɔgɔnna kan. Jeli dɔ nɔgɔlen don ni Coomassie Brilliant Blue (Bio-Rad, UK) ye walasa ka RC-L yɔrɔ fitinin doni ani k’a ye. Protéine min bɛ jeli filanan kan, o bilala polivinilidini fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) la min bɛ baara kɛ ni mɛtanɔli ye (Thermo Fisher Scientific, UK) walasa ka farikolo tangacogo sɛgɛsɛgɛ. PVDF membrane datugulen don 50 mM tris-HCl (pH 7,6), 150 mM NaCl, 0,2% (v / v) Tween-20 ani 5% (w / v) nɔnɔmugu sɔgɔlen na, ka sɔrɔ ka a wuli ni anti-His anticorps fɔlɔ ye (in Dilute the antibody buffer [50 mM tris-HCl (pH 7,6), 150 mM NaCl ani 0,05% (v/v) Tween-20] kɛ 1:1000 A190-114A la, Bethyl Laboratories, Etazuni) la sanga 4 kɔnɔ. Ni aw ye a ko siɲɛ 3 miniti 5 kɔnɔ banakisɛfagalanw tampon na, aw bɛ membrane fara ɲɔgɔn kan ni horseradish peroxidase (Sigma-Aldrich, UK) anti-sosow ka banakisɛfagalan filanan ye (a dilanna 1:10.000 anticorps tampon kɔnɔ) Aw bɛ a bila ka a to a ka se ka a dɔn (miniti 5 ko 3 kɔfɛ anticorps tampon kɔnɔ) ni WESTAR ETA C 2.0 chemiluminescence substrate ye (Cyanagen, Itali) ani Amersham Imager 600 (GE Healthcare, Angletɛri).
Ni aw ye jeli nɔgɔlen walima farikolo tangalan sira kelen-kelen bɛɛ fanga tilatilali ja, ka yɔrɔ min bɛ kulu jukɔrɔ, o fara ɲɔgɔn kan ani ka RC-L (gel nɔgɔlen) ni Protéine-W (immunoassay) fanga hakɛ jateminɛ, ImageJ (57) kɔnɔ Ja in baara. O hakɛw jiginna ka kɛ molari hakɛw ye, k’a miiri ko RC-L ni poroteyini-W hakɛ min bɛ RC-LH114-W sɛgɛsɛgɛli saniyalen na, o ye 1:1 ye ani ka kunnafonidilan bɛɛ normalize ka kɛɲɛ n’o ye.
Walasa ka kunnafoni wɛrɛw sɔrɔ nin barokun in kama, aw bɛ se ka http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1 lajɛ
Nin ye dakun dafalen ye min bɛ tilatila Creative Commons Attribution License sariyaw kɔnɔ. Barokun bɛ sira di baarakɛcogo ma, a tilatilali ani a lasegincogo ma dan tɛ min na, ni a kɛra cogo o cogo, ni baara fɔlɔ fɔra ka ɲɛ.
Kɔlɔsili: An b’a ɲini i fɛ dɔrɔn i k’i ka e-mail ladɛrɛsi di walasa i bɛ mɔgɔ min laadi ɲɛ kan, o k’a dɔn ko i b’a fɛ u ka o e-mail ye, wa ko o tɛ spam ye. An tɛna imɛri ladɛrɛsi si minɛ.
Nin ɲininkali in bɛ kɛ walasa k’a lajɛ n’i ye taamakɛla ye ani ka spam ci otomatiki bali.
David J. K. Swainsbury, Park Qian, Philip J. Jakson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabɛti C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniɛl P Canniffe , Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Yeelen-jaan 1-komplekisi min bɛ farikoloɲɛnajɛ-yɔrɔ la, o jɔcogo min ka bon kosɛbɛ, o bɛ hakilina kuraw di kinone dinamiki kan.
David J. K. Swainsbury, Park Qian, Philip J. Jakson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabɛti C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniɛl P Canniffe , Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Yeelen-jaan 1-komplekisi min bɛ farikoloɲɛnajɛ-yɔrɔ la, o jɔcogo min ka bon kosɛbɛ, o bɛ hakilina kuraw di kinone dinamiki kan.
©2021 Ameriki ka dɔnniya yiriwali tɔnba. jo bɛɛ bɛ a bolo. AAAS ye HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef ani COUNTER ka jɛɲɔgɔn ye. Dɔnniya ɲɛtaa ISSN 2375-2548.