Amrɔ nɛɛ†Adrɛs ni yɔɔ amrɔ nɛɛ: OX11 0DE, UK, Diamond Tsu lɛ, Harwell Jeŋ Nilee kɛ Nibii Heei Afeemɔ He, Dietcote, Oxfordshire, UK, Diamond La He Nitsumɔhe lɛ, Ltd.
Nɔ ni haa kane lɛ feɔ ekome 1 (RC-LH1) ji nɔ titri ni haa tsɛŋemɔ hela ni ji purple fototrofik bacteria lɛ tsuɔ nii. Wɔkɛ RC-LH1 ni jɛ Rhodopseudomonas palustris mli lɛ mli nibii bibii ni akɛkwɛɔ nii amli lɛ enyɔ ba. 2.65-Å yiŋkpɛɛ gbɛjianɔtoo ni yɔɔ RC-LH114-W tsɔne lɛ mli lɛ yɛ 14 subunit LH1 loops ni ebɔle RC he, ni protein W tsiɔ naa, yɛ be mli ni tsɔne ni protein-W bɛ mli lɛ ji RC tsɔne ni RC ebɔle ehe kwraa. LH1 loop lɛ mli fãi 16 lɛ agbe naa. Nibii nɛɛ ni akɛtoɔ he lɛ haa wɔleɔ bɔ ni quinone lɛ tsuɔ nii ehaa yɛ RC-LH1 complex lɛ mli, ni nɔ ni fata he ji tsakemɔi ni atsɔɔɔ mli tsutsu lɛ beni akɛ quinone fĩɔ RC QB he lɛ, kɛ agbɛnɛ hu he ni yelikɛbuamɔ quinone fĩɔ hei lɛ yɔɔ, ni yeɔ ebuaa ni akɛ amɛ yaa RC. W protein lɛ gbɛjianɔtoo krɛdɛɛ lɛ tsiɔ LH1 loop lɛ naa, ni no haa gbɛ ko baa ni atsɔɔ nɔ ahaa quinone/quinolone tsakemɔ lɛ yaa oya.
Hewalɛ ni fotosintɛsis haa lɛ baanyɛ aha wala fɛɛ ni yɔɔ shikpɔŋ lɛ nɔ lɛ ahi shi, ni eyɛ nyɛmɔ babaoo kɛha hulu nibii ahe nilee. Yɛ be mli ni ewoɔ jeŋ muu fɛɛ fotosintɛsis he hewalɛ lɛ, purple fototrofik bacteria hu jieɔ hewalɛ gbɛi srɔtoi kɛ nibii ni haa mɔ he jɔɔ lɛ ahe nyɛmɔi akpo. Amɛbaanyɛ amɛtsi amɛhe kɛjɛ fotosintɛsis he ni amɛdara akɛ heterotrofik bacteria yɛ duŋ mli, amɛbaanyɛ amɛsaa nitrogen kɛ carbon dioxide, amɛfee hydrogen, ni amɛfite nibii ni haa mɔ he jɔɔ lɛ (1-3). Bɔni afee ni aná hewalɛ kɛha nifeemɔi nɛɛ, esa akɛ atsake la lɛ oya ni efee tsofa hewalɛ. Nɛkɛ gbɛ nɛɛ jeɔ shishi beni antenna ni mɔɔ la lɛ ŋmɛɔ la ni ekɛ hewalɛ ni mɔmɔ lɛ lɛ yaa he ni nibii lɛ feɔ lɛ (RC) lɛ, ni no haa charge lɛ mligbalamɔ jeɔ shishi (4 – 7). Fotosintɛsis shishijee kuu ni yɔɔ purple fototrofik bacteria mli lɛ ji RC henɔ 2, ni kane-kpamɔ nibii 1 (LH1) ebɔle he, ni feɔ RC-LH1 core complex lɛ. LH1 lɛ ji nɔ ni αβ heterodimers ni ebɔle ehe lɛ feɔ, ni eko fɛɛ eko kɛ bacteria klorofil (BChl) enyɔ ni ji molecules kɛ carotenoids kome loo enyɔ (8-12) lɛ feɔ ekome. LH1 antenna ni yɔɔ mlɛo fe fɛɛ lɛ ji 16 loo 17 αβ heterodimers ni bɔleɔ RC (9-13) he yɛ ŋwɛi ŋta ni agbe naa mli, shi yɛ shishijee nibii krokomɛi amli lɛ, transmembrane peptides lɛ gbalaa LH1 ni ebɔle lɛ lɛ nɔyaa lɛ naa, No hewɔ lɛ, ehaa quinol/quinone gbɛfaŋnɔ ni yɔɔ bRC, cytoch1c lɛ teŋ lɛ yaa hiɛ. 13-15 lɛ nɔ). Tso ni ji purple fototrofik Rhodopseudomonas (Rps.) ji nɔkwɛmɔnɔ ni baanyɛ anu hewalɛ kɛ electron tsɔne ni fiɔ fotosintɛsis sɛɛ lɛ shishi. Klɛŋklɛŋ kristalo shishitoo mla ni ji Rps. Nɔkwɛmɔnɔ ni kɔɔ palustris RC-LH1 he lɛ ji RC, ni heterodimeric LH1 ŋmeii 15 ebɔle he, ni protein ko ni aleee ni atsɛɔ lɛ “Protein W” (14) lɛ tsiɔ amɛnaa. No sɛɛ lɛ, ana Protein-W akɛ RPA4402, ni ji 10.5kDa protein ni bɛ su ko ni yɔɔ helices etɛ ni atsɔɔ hiɛ atsɔɔ akɛ ebaatsɔ mli (TMH) (16). Wɔkɛɔ akɛ wɔbaatsake rpa4402 tsɛŋemɔ hela ni kɛ protein W woɔ mli lɛ gbɛi wɔwo pufW koni ekɛ gbɛi ni akɛtsuɔ nii kɛhaa tsɛŋemɔ helai ni kɛ RC-L, M (pufL, pufM) kɛ LH1α, β (pufA, pufB) fãi bibii lɛ kpãa gbee. Nɔ ni yɔɔ miishɛɛ ji akɛ, protein-W lɛ yɛ aaafee RC-LH1 lɛ mlijaa 10 pɛ mli, ni ejieɔ Rps kpo. palustris lɛ feɔ RC-LH1 srɔtoi enyɔ. Yɛ biɛ lɛ, wɔbɔɔ ŋwɛi ŋtayaai enyɔ ni yɔɔ ŋtayaai amli lɛ ahe amaniɛ, eko yɛ protein W kɛ 14 αβ heterodimers, ekroko lɛ bɛ ŋtayaai W kɛ 16 Heterodimer LH1 loop ni agbe naa lɛ. Wɔ gbɛjianɔtoo lɛ damɔ shi kɛha tsakemɔ ko ni ba yɛ RC-LH1 complex ni ji Rps lɛ shishinumɔ mli. palustris, ejaakɛ wɔpɛi gbɔmɛi ayibɔ ni yɔɔ srɔtoi lɛ eko fɛɛ eko mli lɛ mli ni wɔyɛ yiŋkpɛɛ ni fa bɔ ni sa ni wɔkɛbaahã peptide fɛɛ peptide kɛ pigments ni akɛfiɔ he kɛ lipids kɛ quinones ni kɔɔ he lɛ yɛ faŋŋ mli. Nibii nɛɛ ni akɛtoɔ he lɛ tsɔɔ akɛ TMH proteins-W etɛ ni anako yɛ RC-LH1 complex kroko mli kɛbashi amrɔ nɛɛ feɔ quinone gbɛ ko ni haa quinone/quinolone tsakemɔ lɛ yaa oya. Ayoo lipid kɛ quinone ni akɛtoɔ he lɛ ayibɔ, ni wɔjie tsakemɔ hee ko kpo yɛ quinone kɛ RC ni akɛfee ekome lɛ sɛɛ, ni baanyɛ afee nɔ ni sa kɛha fotosistɛm II (PSII) RC ni ji oxygen fototrophic nibii ni wala yɔɔ amɛmli lɛ. Nibii ni wɔna lɛ haa wɔleɔ bɔ ni quinone/quinolone lɛ tsakeɔ amɛhe ehaa yɛ RC-LH1 core complex ni ji purple fototrofik bacteria lɛ mli lɛ he.
Bɔni afee ni akase nibii enyɔ ni anaa yɛ Rps. palustris lɛ, wɔjieɔ RC-LH1 lɛ eko fɛɛ eko kɛjɛɔ he kɛtsɔ tsofafeemɔ gbɛ̀i anɔ. Atsumɔ protein W-deficient complex (ni sɛɛ mli lɛ atsɛɔ lɛ ΔpufW) lɛ kɛjɛ tsɛŋemɔ hela ni bɛ pufW tsɛŋemɔ hela lɛ mli (16), ni RC-LH1 tsɛŋemɔ hela kome pɛ ji nɔ ni abaanyɛ afee. Protein W-ni yɔɔ mli lɛ ji nɔ ni tsɛŋemɔ hela ko feɔ. Atsakeɔ protein W ni yɔɔ nɛkɛ tsofa nɛɛ mli lɛ kɛ 10x His tag yɛ e-C-terminus lɛ nɔ, koni anyɛ akɛ protein W ni yɔɔ complex lɛ mli lɛ afata protein W ni bɛ mli lɛ babaoo he jogbaŋŋ kɛtsɔ dade ni haaa enyɛ etsu nii lɛ nɔ. Akɛ nɔ ni yɔɔ srɔtoi lɛ mli gbalaa jogbaŋŋ (16) Affinity Kromatograf (IMAC).
Taakɛ atsɔɔ yɛ mfoniri 1 lɛ mli lɛ, RC (RC-L, RC-M kɛ RC-H) ni ji fãi bibii etɛ ni LH1 antenna ebɔle amɛhe lɛ yɛ ŋtayaai enyɔ lɛ fɛɛ mli. 2.80-A gbɛjianɔtoo ni yɔɔ tsɔne ni bɛ protein-W lɛ mli lɛ tsɔɔ αβ heterodimers 16, ni feɔ LH1 loop ni agbe naa ni ebɔle RC he kwraa, ni sɛɛ mli lɛ atsɛɔ lɛ RC-LH116 tsɔne lɛ. 2.65Å ni yɔɔ protein-W-ni yɔɔ mli lɛ mli lɛ yɛ 14-heterodimer LH1 ni protein-W lɛ kpata mli, ni sɛɛ mli lɛ atsɛɔ lɛ RC-LH114-W.
(A kɛ B) Nɔ ni damɔ shi kɛha nɔ ni akɛfee lɛ. (C kɛ D) Pigments ni akɛfee ekome ni ajieɔ lɛ kpo yɛ tso mli. (E kɛ F) Nibii ni yɔɔ srɔtoi ni ana kɛjɛɔ cytoplasmic lɛ hiɛ lɛ yɛ peptides kɛ LH1 subunits ni ajieɔ lɛ kpo yɛ mfonirifeemɔ mli lɛ, ni awoɔ amɛ yibɔi yɛ ŋmɛlɛtswaa gbɛ nɔ kɛjɛɔ protein-W gap lɛ mli [ni kɛ Rba yibɔi lɛ kpãa gbee. spɛroidɛs ni mli wa (13)]. Kɛha LH1-α lɛ, protein lɛ mli fã bibioo lɛ su ji ŋmɔŋ; kɛha LH1-β lɛ, protein lɛ mli fã bibioo lɛ su ji bluu; kɛha protein-W lɛ, protein lɛ yɛ tsuru; kɛha RC-H lɛ, eji cyan; kɛha RC-L lɛ, eji Orange; kɛha RC-M, ŋmɔlɔŋmɔlɔ. Akɛ tso damɔ shi kɛha cofactors, eŋɔli damɔ shi kɛha BChl kɛ BPh a molekulii, purple damɔ shi kɛha carotenoids, ni ŋmɔŋ damɔ shi kɛha UQ10 molekulii. (G kɛ H) Protein-W gap ni yɔɔ RC-LH114-W (G) kɛ RC-LH116 (H) lɛ kpokpaa ni tamɔ amɛhe lɛ nɔ lɛ he mfoniri ni ajie lɛ kpo. Ajieɔ cofactors lɛ kpo yɛ gbɛ ni haa ŋwɛi ŋta lɛ mli woɔ mli lɛ nɔ, ajieɔ chelated quinone lɛ kpo yɛ bluu mli. Akɛ bluu ni yɔɔ (G) lɛ maa protein-W gap lɛ nɔ mi, ni buji bibii ni quinone/quinolol gbɛɔ eshwaa yɛ LH116 ring lɛ nɔ lɛ ji nɔ ni akɛ bluu ni yɔɔ (H) lɛ maa nɔ mi.
(A kɛ B) tsɔɔ RC ni LH1αβ heterodimers ni agbele loo agbele mli lɛ ebɔle ehe, ni eko fɛɛ eko fiɔ BChl enyɔ kɛ carotenoid kome he (Mfoniri 1, C kɛ D). Nibii amlipɛimɔi ni eho lɛ etsɔɔ akɛ Rps ji LH1 complex lɛ. Yɛ spirulina xanthin lɛ gbɛ ni atsɔɔ nɔ afeɔ nibii ni wala yɔɔ mli lɛ mli lɛ, nɛkɛ kooloi srɔtoi nɛɛ yɛ carotenoids ni efutu (17). Shi kɛlɛ, spiropyrroxanthin ji nɔ ni fe fɛɛ ni haa mɔ tsui nyɔɔ emli, ni egbɛjianɔtoo lɛ haa mɔ tsui nyɔɔ emli. No hewɔ lɛ, wɔhala akɛ wɔbaafee spiroxanthin he nɔkwɛmɔnɔ yɛ LH1 ni akɛkpɛtɛɔ he lɛ fɛɛ mli. Alfa kɛ beta polypeptides lɛ ji TMH komekomei ni yɔɔ kponɔgbɛ fãi kukuji (Mfoniri 1, A, B, E, kɛ F). Eyɛ mli akɛ anaaa bɔ ni 17 ni eshwɛ yɛ C-terminus lɛ mli lɛ fa ha moŋ, shi ajie alpha polypeptide lɛ kɛjɛ Met1 kɛtee Ala46 yɛ fãi enyɔ lɛ fɛɛ mli. 116, kɛjɛ Ser5 kɛtee Tyr52 yɛ RC-LH114-W mli. loo 4 N-terminal loo 13 C-terminal nibii ni eshwɛ lɛ ayibɔ ko kwraa bɛ (Mfoniri S1). RC-LH1 ni akɛfutu ni asaa kɛjɛ koo mli tsɛŋemɔ hela lɛ mli lɛ mlipɛimɔ tsɔɔ akɛ kpokpaa ni elaaje lɛ ji nɔ ni jɛ nɛkɛ peptides nɛɛ mligbalamɔ ni yɔɔ srɔtoi lɛ mli ba (Mfoniri S1 kɛ S2). Akwɛ N-naagbee formylation ni ji α-Met1 lɛ hu (f). Nibii amlipɛimɔ lɛ tsɔɔ akɛ α-peptide lɛ mli nibii ni eshwɛ lɛ ji fMet1 kɛyashi Asp42/Ala46/Ala47/Ala50, ni β-peptide lɛ mli nibii ni eshwɛ ji Ser2 kɛyashi Ala53, ni enɛ kɛ EM density map ni hulutsoo be lɛ baa shi lɛ kpãa gbee jogbaŋŋ.
α-His29 kɛ β-His36 ni feɔ ekome lɛ haa BChls feɔ hiɛ kɛ hiɛ; αβ heterodimer fɛɛ heterodimer kɛ ekutsoŋbii lɛ buaa amɛhe naa koni amɛfee ŋwɛi ŋta ni agbele naa (RC-LH114-W) loo ŋta ni agbele naa (RC-LH116) ni bɔleɔ RC lɛ he Exciton ni kɛ pigment ni yɔɔ mli lɛ feɔ ekome (Mfoniri 1, C kɛ D). Kɛ akɛto 877 nm band ni ji RC-LH114-W lɛ he lɛ, 880 nm ni ŋmɛɔ red shift ni ji RC-LH116 lɛ ji 3 nm (Mfoniri 2A). Shi kɛlɛ, circular dichroism spectrum lɛ miihe afee ekome (Mfoniri 2B), ni tsɔɔ akɛ, eyɛ mli akɛ srɔtofeemɔ ko ni yɔɔ faŋŋ yɛ loops ni agbele kɛ loops ni agbele lɛ ateŋ moŋ, shi BChls lɛ akutso lɛ mli shihilɛ lɛ tamɔ amɛhe waa. Nɔ ni jɛɔ redshift ni ajieɔ kɛjɛɔ mli lɛ mli baa ji hulutsoo ni baa shi kɛ shifimɔ ni yaa hiɛ yɛ ŋwɛi ŋta ni agbe naa lɛ nɔ (18, 19), tsakemɔ ni baa yɛ ŋta ni agbe naa lɛ mli lɛ mli (20, 21), loo nɛkɛ nibii enyɔ nɛɛ ni akɛfee ekome (11).
(A) Ultraviolet/ni anaa/ni bɛŋkɛ-infrared ni ŋmɛɔ la, ni akɛ amɛhe nibii ni kɛ amɛhe kpãa gbee lɛ kadiɔ amɛyitsei lɛ ni afee amɛ bɔ ni sa kɛyashɛɔ BPh yiteŋgbɛ ni yɔɔ 775 nm lɛ nɔ. (B) Dichroism ŋwɛi ŋta ni yɔɔ ŋwɛi ŋta ni ji BChl ni shɛɔ 805 nm. (C kɛ D) Ahala ΔA ŋwɛibɔfoi kɛjɛ be-ni-atsuɔ he nii ni RC-LH114-W ŋwɛibɔfoi (C) kɛ RC-LH116 ŋwɛibɔfoi (D) lɛ mli. Kɛha nɔ ni akɛtoɔ he jogbaŋŋ lɛ, spectra fɛɛ ji nɔ ni sa kɛyaa ∆A ni ji −A yɛ 0.2 ps. (E) Bɔ ni cytochrome c2 oxidation lɛ yaa nɔ ehaa yɛ irradiation sɛɛ yɛ UQ2 mli nibii srɔtoi ni yɔɔ mli lɛ ahiɛ (kwɛmɔ mfoniri S8 kɛha saji ni atsɔɔ mli lɛ). (F) Yɛ wala yibii ni dara yɛ la ni mli wa fioo, teŋgbɛ loo la ni mli wa waa shishi (10, 30 loo 300μMm-2 s-1, yɛ gbɛ kome nɔ lɛ), protein W kɛ RC-L subunits ni yɔɔ tsɔne ni atsuu he lɛ mli lɛ kɛ tsɔne ni mli egbala lɛ mlijaa lɛ. Tsɔɔmɔ niyenii ni haa mɔ he waa lɛ falɛ kɛtsɔ SDS-polyacrylamide gel tsɔne ni akɛkwɛɔ nibii amli kɛ tsofa ni haa mɔ he waa lɛ nɔ (kwɛmɔ mfoniri S9 kɛha saji ni aŋmalako). Kwɛmɔ bɔ ni RC-LH114-W ni atsuu he lɛ yɔɔ ha lɛ. Stoikiomɛtrik mlijaa ni yɔɔ RC-L kɛ protein-W ni yɔɔ mli lɛ ji 1:1.
BChls ni yɔɔ gbɛhe 1 yɛ RC-LH114-W (Mfoniri 1, A, C, kɛ E) αβ14 loop ni efite lɛ mli lɛ bɛŋkɛ RC shishijee shika halɔ (P) lɛ he kɛ 6.8Å fe BChls ni tamɔ nakai ni yɔɔ RC-LH116 (Mfoniri. 1, B, S3, D); shi kɛlɛ, be kukuoo mli nibii ni haa mɔ tsui nyɔɔ emli lɛ tsɔɔ akɛ, kɛha RC-LH114-W kɛ RC-LH116 lɛ, sɛɛkuu hewalɛ ni ajieɔ kɛjɛɔ LH1 kɛyaa RC lɛ mli lɛ ji 40 ±4 kɛ 44±3 ps (Mfoniri 2). , C kɛ D, Mfoniri S4 kɛ Okadi S2). Srɔtofeemɔ ko kwraa bɛ tsɔne ni akɛyaa he kroko yɛ RC mli lɛ hu mli (Mfoniri S5 kɛ niŋmaa ni fata he ni kɔɔ he lɛ). Wɔsusuɔ akɛ hewalɛ ni akɛhaa lɛ be ni bɛŋkɛ kpaakpa ni ka LH1 kɛ RC-P teŋ lɛ jɛɔ sɛɛjɛkɛmɔ, ŋwɛigbɛ kɛ hewalɛ ni baanyɛ aba ni BChl babaoo yɔɔ LH1 loops enyɔ lɛ mli lɛ hewɔ. Etamɔ nɔ ni LH1 hewalɛ gbɛjianɔtoo lɛ mlitaomɔ koni ashɛ he ni jɛkɛmɔ ni baa shi fe fɛɛ lɛ he lɛ eyaaa oya fe hewalɛ ni ajieɔ kɛjɛɔ hei ni hi jogbaŋŋ kɛyaa RC lɛ tɛ̃ɛ. LH1 loop ni yɔɔ RC-LH114-W lɛ hu baanyɛ atsɔ dɔlɛ ni he ehiaaa yɛ dɔlɛ shihilɛi ni baa shi mli kɛha gbɛjianɔtoo mlipɛimɔ, ni αβ14 ring ni kɛlɛ yɛ tsu mli dɔlɛ mli kɛjɛ βBChls ni yɔɔ RC 1 lɛ he lɛ.
RC-LH116 complex lɛ mli BChls 32 kɛ carotenoids 16 yɔɔ mli, ni egbɛjianɔtoo fɛɛ lɛ tamɔ nɔ ni anáa kɛjɛɔ Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S) (9), Thiorhodovibrio ID9T9in (9), Thiorhodovibrio ID9T9r. (12) kɛ ŋmeii ni yɔɔ ŋmɔji amli (Blc.viridis) (PDB ID 6ET5) (10). Yɛ be mli ni ato he gbɛjianɔ lɛ sɛɛ lɛ, ana sɛɛkuu bibii pɛ yɛ αβ heterodimers lɛ agbɛhei lɛ amli, titri lɛ 1-5, 15, kɛ 16 (Mfoniri S6). Protein-W ni yɔɔ lɛ náa LH1 lɛ nɔ hewalɛ waa. E TMH etɛ ɔmɛ a ngɔ ngmɔku kukuji kɛ tsa, nɛ N-terminal ɔ ngɛ lumen fã nɛ ngɛ complex ɔ mi ɔ, nɛ C-terminal ɔ hu ngɛ cytoplasmic blɔ nya tomi ɔ nɔ (Mfoniri 1A kɛ 3, A kɛ ya si D). Protein-W lɛ ji nɔ ni sumɔɔɔ nu babaoo (Mfoniri 3B), ni TMH2 kɛ TMH3 kɛ LH1αβ-14 feɔ ekome ni amɛfeɔ transmembrane hiɛ (Mfoniri 3, B kɛ E kɛyashi G). Phe, Leu kɛ Val nibii ni eshwɛ yɛ transmembrane kpokpaa lɛ nɔ lɛ ji nɔ titri ni yɔɔ mli. Akɛ amino acids ni haaa nu mli kɛ αβ-14 ni haa mɔ he jɔɔ lɛ eto nɛkɛ nibii ni eshwɛ nɛɛ ahe. Polar nibii ni eshwɛ lɛ ekomɛi hu yeɔ ebuaa ni amɛfeɔ ekome, ni nɔ ni fata he ji hydrogen ni yɔɔ W-Thr68 kɛ β-Trp42 teŋ ni yɔɔ ŋwɛi ŋta ni mli wa lɛ hiɛ lɛ (Mfoniri 3, F kɛ G). Yɛ tsɛŋemɔ hela lɛ hiɛ lɛ, Gln34 lɛ bɛŋkɛɔ keto kuu ni ji αβ-14 carotenoids lɛ. Kɛfata he lɛ, atsu n-dodecyl β-d-maltoside (β-DDM) lɛ he nii, ni etsitsi ni nyɛɛɛ nu abote mli lɛ tee nɔ eshɛ he ni protein-W kɛ αβ-14 teŋ lɛ yɔɔ lɛ, ni ekolɛ lipid tsitsi lɛ yɛ gbɔmɔtso lɛ mli. Wɔyɔse hu akɛ C-terminal resolution kpokpai ni yɔɔ protein W kɛ RCH mli lɛ bɛŋkɛ kpaakpa, shi jeee yɛ bɔ ni afee nibii pɔtɛɛ komɛi ni kɛ amɛhe feɔ ekome lɛ mli (Mfoniri 1, A kɛ E). Shi kɛlɛ, ekolɛ tsakpaa ko yɛ C-terminal amino acids ni asaaa he yɛ nɛkɛ protein enyɔ nɛɛ amli lɛ mli, ni baanyɛ aha gbɛ ko aba kɛha protein-W lɛ nitsumɔ yɛ RC-LH114-W complex lɛ kpee lɛ mli.
(A) Protein-W, ni kɛ LH1αβ14 kpeɔ yɛ kaatoo mli lɛ, yɛ sɛɛ ŋwɛi ŋta ni tamɔ tso (tsuru), ni ajieɔ lɛ kpo yɛ electrostatic potential diagram lɛ fã ko mli (ŋwɛi ŋta ni mli tse ni ŋwɛi ŋta ni ji 0.13). (B) Protein-W ni damɔ shi kɛha nɔ ko ni yɔɔ su ni nu bɛ mli lɛ. Ajieɔ hei ni yɔɔ polar kɛ charged lɛ akpo yɛ cyan mli, ajieɔ hei ni hydrophobic yɔɔ lɛ kpo yɛ eyɛŋ mli, ni ajieɔ hei ni hydrophobic yɔɔ mli waa lɛ akpo yɛ orange mli. (C kɛ D) Protein-W ni ajie lɛ kpo yɛ kaatoo mli lɛ, egbɛtsɔɔmɔ lɛ tamɔ nɔ ni yɔɔ (A) (C) lɛ nɔŋŋ, ni atsakeɔ lɛ 180° (D). Taakɛ gbɛhe ni yɔɔ sɛɛkuu lɛ mli lɛ tsɔɔ lɛ, nibii ni eshwɛ ni anyɛɔ ayɔseɔ amɛ lɛ kɛ ŋwɛibɔfo su ko tsuɔ nii, he ni N-naagbee lɛ ji bluu ni C-naagbee lɛ ji etsuru. (E) Protein-W yɛ susumɔ kome mli tamɔ nɔ ni yɔɔ (A) lɛ mli, ni nɔ ni eshwɛ yɛ protein-W:LH1 teŋ lɛ damɔ shi kɛha tso ni akɛ okadii ekpɛtɛ he. (F) Protein-W lɛ tsɔɔ 90° kɛyaa (E) kɛ LH1αβ14 nɔ yɛ kaatoo najiaŋdamɔlɔ lɛ mli, kɛ nɔ ni kɔɔ sɛɛgbɛ nibii ni eshwɛ yɛ bar najiaŋdamɔlɔ lɛ mli lɛ he. Akɛ nibii ni eshwɛ ni jɛɔ beta polypeptide lɛ mli lɛ woɔ nɔ. Atsɔɔ cofactor lɛ akɛ bar ni kɛ Mfoniri 1 lɛ su lɛ kpãa gbee, ajieɔ β-DDM ni efite lɛ kpo yɛ ŋmɔtɔ mli, ni atsɔɔ oxygen lɛ yɛ ŋmɔtɔ mli. (G) Atsake nɔ ni anaa yɛ (F) lɛ mli lɛ 180°, ni alpha polypeptide ni aŋmala nɔ lɛ mli nibii ni eshwɛ ni jeɔ kpo faŋŋ lɛ yɔɔ mli.
Protein-W yeɔ αβ heterodimer (ni ji 15 yɛ mfoniri 1F lɛ mli) najiaŋ, ni no haaa loop lɛ agbe naa ni ekɛ klɛŋklɛŋ αβ heterodimers etɛ lɛ shwieɔ shi. A na kaa kekleekle αβ-1 heterodimer ɔ hɛ mi nyami nɛ pe kulaa ngɛ sini ɔ mi ɔ ji 25° kɛ ya si 29° (Mfoniri 1, A kɛ E), nɛ 2° kɛ ya si 8° hɛ mi nyami nɛ αβ-1 ngɛ RC A sharp contrast-LH116 kɛ F (Mfoniri 1, B) ɔ pee. Heterodimers ni ji enyɔ kɛ etɛ lɛ shwieɔ shi yɛ 12° kɛyashi 22° kɛ 5° kɛyashi 10°, yɛ amɛteŋ. Yɛ RC he gbɛtsii nii lɛ hewɔ lɛ, αβ-1 lɛ sɛɛkuu lɛ fataaa αβ ni ji enyɔ lɛ he (ni kɛ αβ ni ji 16 lɛ kpãa gbee yɛ mfoniri 1F lɛ mli), no hewɔ lɛ ehaa gbɛ ni yɔɔ faŋŋ baa LH1 ring lɛ mli (mfoniri 1, A kɛ E) . Akɛni αβ heterodimers enyɔ bɛ, ni BChl ejwɛ kɛ carotenoids enyɔ ni laajeɔ lɛ fata he hewɔ lɛ, carotenoids lɛ eko kwraa kɛ αβ-1 subunit ni etsɔ mli lɛ kpaaa gbee, ni no haa LH114-W ring ni carotenoids 13 Vegetarian kɛ BChls 28 yɔɔ mli lɛ baa. Nibii ni yɔɔ αβ1 kɛyashi 7 lɛ amli lɛ baa shi fe LH1 loop lɛ fã kroko lɛ, ni baanyɛ ajie plasticity ni yɔɔ LH1 subunit ni bɛŋkɛ RC QB he lɛ kpo (Mfoniri 4) .
Atsɔɔ RC-LH114-W (A kɛ B) kɛ RC-LH116 (C kɛ D) mfonirii lɛ kɛjɛɔ ŋwɛi kwɛmɔ/sɛɛ kwɛmɔ (A kɛ B) (A kɛ C) kɛ ŋwɛi ŋta ni yɔɔ Fig. 1. (B kɛ D) lɛ nɔŋŋ mli. Atsɔɔ saji ni yɔɔ kɔla lɛ yɛ ninejurɔgbɛ.
1:14 ji Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Shi kɛlɛ, protein W kɛ PufX bɛ nɔ ko ni yɔɔ faŋŋ ni tamɔ amɛhe, ni amɛyɛ hewalɛ ni sa kadimɔ waa yɛ amɛ LH1 gbɛjianɔtoi lɛ anɔ. PufX ji TMH kome ni yɔɔ N-naagbee tsɔne ni kɛ RC-H subunit (13) lɛ fã lɛ tsuɔ nii yɛ he ko ni kɛ Rps kpãa gbee. tsɛŋemɔ hela LH116αβ-16. PufX feɔ gbɛ kɛha quinone/quinolone tsakemɔ ni yɔɔ RC-LH1 kɛ cytochrome bcl complex lɛ teŋ lɛ ni eyɛ Rba fɛɛ mli. sfaeroides shishitoo mla (13). Eyɛ mli akɛ monomer-monomer teŋgbɛ lɛ yɛ Rba mli moŋ. Sphaeroides RC-LH1-PufX dimer lɛ yɛ he ni protein W ni yɔɔ RC-LH14-W lɛ mli lɛ kpãa gbee yɛ lɛ, ni gbɛ ni PufX kɛ protein-W lɛ kɛbaa lɛ yɛ he ni tamɔ nakai nɔŋŋ (Mfoniri S7A). RC-LH114-W lɛ mli srɔtofeemɔ lɛ hu kɛ susumɔ ni ji quinone gbɛ (8) ni ji Pseudomonas rosea LH1 lɛ kpãa gbee, ni peptides ni kɛ protein W loo PufX bɛ tsakpaa lɛ feɔ lɛ (Mfoniri S7B). Kɛfata he lɛ, quinone gbɛ ni yɔɔ Blc. LH1 ni ji emerald green ni afee kɛtsɔ γ subunit kome ni ajieɔ kɛjɛɔ mli lɛ nɔ (7) lɛ yɛ he ko ni tamɔ nakai nɔŋŋ (Mfoniri S7C). Eyɛ mli akɛ protein sɔrɔtoi ji mɛi ni kɛ amɛhe woɔ mli moŋ, shi etamɔ nɔ ni nɛkɛ quinone/quinolol gbɛi nɛɛ kpojiemɔ yɛ he kome yɛ RC-LH1 complex lɛ mli lɛ ji nɔkwɛmɔnɔ ni tsɔɔ bɔ ni sutsakemɔ ni yaa nɔ ehaa lɛ yaa hiɛ ehaa, ni tsɔɔ akɛ gbɛ ni protein W kɛbaa lɛ baanyɛ atsu nii akɛ quinone gbɛ.
Gbɛ ni kã LH114-W loop lɛ mli lɛ haa anyɛɔ afeɔ membrane kpokpaa ko ni yaa nɔ yɛ RC-LH114-W complex lɛ mligbɛ he lɛ kɛ membrane babaoo lɛ teŋ (Mfoniri 1G), moŋ fe ni akɛ hei enyɔ lɛ baatsa kɛtsɔ protein pore nɔ tamɔ yɛ protein mli lɛ. RC-LH116 tsɔne lɛ tamɔ Tch ni agbe naa lɛ. Nɔ ni tamɔ tsɔne ni akɛwoɔ mɔ mli (22) (Mfoniri 1H). Akɛni quinone ni gbɛɔ eshwaa kɛtsɔɔ membrane lɛ mli lɛ yaa oya fe bɔ ni egbɛɔ eshwaa kɛtsɔɔ protein gbɛ bibioo lɛ nɔ hewɔ lɛ, LH114-W loop ni agbele lɛ baanyɛ aha RC lɛ atsake oya fe LH116 loop ni agbele lɛ, ni quinone ni gbɛɔ eshwaa kɛyaa RC lɛ mli lɛ baanyɛ afee nɔ ni tsii fe tsutsu lɛ. Bɔni afee ni wɔka wɔkwɛ kɛji protein W lɛ saa quinones lɛ tsakemɔ he kɛtsɔɔ RC nɔ lɛ, wɔfee cytochrome oxidation assay ko yɛ ubiquinone 2 (UQ2) (ni ji adebɔɔ mli UQ10 ni yɔɔ isoprene tsitsi kuku lɛ he nɔkwɛmɔnɔ) lɛ nɔ (Mfoniri 2E). Eyɛ mli akɛ chelated quinone ni yɔɔ lɛ tsiɔ Michaelis ni yɔɔ faŋŋ lɛ he yiŋkpɛɛ ni ja lɛ naa moŋ (RC-LH114-W kɛ RC-LH116 sa kɛha 0.2±0.1μM kɛ 0.5±0.2μM, yɛ gbɛ kome nɔ), shi RC-LH±2.RC114-0. 28±5% ni da fe RC-LH116 (3.6±0.1 e-RC-1 s-1).
Shishijee lɛ, wɔbu akɔntaa akɛ protein-W lɛ yɛ aaafee 10% yɛ core complex lɛ mli (16); biɛ lɛ, mɛi ni yɔɔ jɛmɛ lɛ ayibɔ ni ji 15±0.6%, 11±1% kɛ 0.9±0.5, % (Mfoniri 2F). Nibii ni akɛtoɔ mass spectrometry he lɛ tsɔɔ akɛ, histidine tag ni akɛfata he lɛ haaa protein-W ni fa babaoo lɛ aba shi kɛ akɛto koo mli tsɛŋemɔ helai ahe (P = 0.59), no hewɔ lɛ, nɛkɛ ŋwɛi gbɛhei nɛɛ jeee nɔ ko ni akɛfee nibii ni akɛtsake protein-W (Mfoniri S10). Shi kɛlɛ, nɛkɛ protein-W ni yɔɔ RC-LH1 lɛ mli lɛ baanyɛ aha RC komɛi atsɔ oya, ni no baaha quinone/quinolone tsakemɔ ni yaa nɔ blɛoo yɛ RC-LH116 lɛ mli lɛ aba shi. Wɔyɔse akɛ la ni fa babaoo ni yɔɔ mli lɛ kɛ transcriptomics data ni aŋmala nyɛsɛɛ nɛɛ kpãaa gbee, ni tsɔɔ akɛ pufW gene kpojiemɔ lɛ yaa hiɛ yɛ la ni naa wa shishi (Mfoniri S11) (23). Srɔtofeemɔ ni yɔɔ pufW niŋmaa kɛ protein-W ni akɛwoɔ RC-LH1 mli lɛ teŋ lɛ ji nɔ ni futuɔ mɔ ni ebaanyɛ ejie protein lɛ he gbɛjianɔtoo ni mli wa lɛ kpo.
Yɛ RC-LH114-W mli lɛ, akɛ tsofai ni haa mɔ tsui nyɔɔ emli 6 (CDL), tsofai ni haa mɔ tsui nyɔɔ emli 7 (POPC), tsofai ni haa mɔ tsui nyɔɔ emli 1 (POPG) kɛ β-DDM 29 haa ni afee amɛ nɔkwɛmɔnɔ yɛ mli. RC-LH116 (Mfoniri 5, A kɛ B). Yɛ nɛkɛ gbɛjianɔtoi enyɔ nɛɛ amli lɛ, eshwɛ fioo ni CDL lɛ baahi cytoplasmic fã ni yɔɔ complex lɛ mli lɛ nɔ, yɛ be mli ni POPC, POPG kɛ β-DDM lɛ babaoo yɛ luminal fã lɛ nɔ. Ajie lipid kɛ detergent molecules enyɔ kɛjɛ αβ-1 kɛyashi αβ-6 kpokpaa ni yɔɔ RC-LH114-W complex lɛ mli (Mfoniri 5A), ni ajie enumɔ kɛjɛ RC-LH116 kpokpaa ni tamɔ nakai lɛ nɔ (Mfoniri 5B ). Ana lipids babaoo yɛ complex lɛ fa kroko lɛ nɔ, titri lɛ CDL, ni abua naa yɛ RC kɛ αβ-7 kɛyashi αβ-13 teŋ (Mfoniri 5, A kɛ B). Lipids kɛ detergents krokomɛi ni atsu he nii yɛ gbɛjianɔtoo naa lɛ yɛ LH1 ring lɛ kponɔ, ni acyl chains ni atsu he nii jogbaŋŋ lɛ yaa hiɛ yɛ LH1 subunits lɛ ateŋ, ni atsɔɔ mli yɛ be kukuoo mli akɛ β-DDM yɛ RC-LH114-W mli, ni atsɔɔ shishi akɛ β-DDM yɛ RC A ni akɛ βPOPC-DHDM kɛ β166 efutu mli. Nibii ni tamɔ nakai ni chelating lipids kɛ detergents yɔɔ wɔ gbɛjianɔtoo lɛ mli lɛ tsɔɔ akɛ amɛji gbɔmɔtsoŋ nibii ni kɔɔ he ni akɛfiɔ he (Mfoniri S12A). Molecules ni tamɔ amɛhe lɛ agbɛhei yɛ Tch mli lɛ hu yɛ tsakpaa kpakpa. Blɛoo kɛ Trv. 970 RC-LH1s (Mfoniri S12, B kɛyashi E) (9, 12) kɛ hydrogen-bonding residues ni yɔɔ lipid yitso kuu lɛ mli lɛ jie sɛɛfimɔ kpakpa kpo yɛ sɛɛkuu mli gbɛjianɔtoo lɛ mli (Mfoniri S13), ni tsɔɔ akɛ Conserved CDL ni kɛ RC (24) lɛ kpãa gbee lɛ, nɛkɛ hei nɛɛ baanyɛ afee comp- HRCx ni ato he gbɛjianɔ.
(A kɛ B) Akɛ mfonirifeemɔi damɔ shi kɛha RC-LH114-W (A) kɛ RC-LH116 (B) peptides lɛ, ni akɛ tsoi damɔ shi kɛha pigments lɛ, ni akɛ su ni yɔɔ Mfoniri 1 lɛ mli lɛ tsuɔ nii. UQ ni akɛfiɔ RC QA kɛ QB hei lɛ ji ŋmɔŋ, yɛ be mli ni UQ ni yɔɔ he kroko lɛ ji bluu. (C kɛ D) Susumɔi ni yɔɔ (A) kɛ (B) lɛ nɔŋŋ, ni ajie lipids kɛjɛ mli. (E kɛyashi G) Q1(E), Q2(F) kɛ Q3(G) ni ajie kɛjɛ RC-LH116 lɛ mli lɛ mlilɛɛmɔ, kɛ sɛɛ ŋwɛi ŋwɛi ni náa amɛhe nɔ hewalɛ. Atsɔɔ hydrogen bonds lɛ akɛ line diji ni yɔɔ ŋmɔlɔŋmɔlɔ.
Yɛ RC-LH116 mli lɛ, RC QA kɛ QB UQ fɛɛ, ni kɛ amɛhe woɔ electron tsɔne mli yɛ charge mligbalamɔ gbɛjianɔtoo lɛ mli lɛ, fiteɔ yɛ amɛhe ni amɛfiɔ amɛhe lɛ. Shi, yɛ RC-LH114-W lɛ mli lɛ, atsuuu QB quinone he nii ni abaawie he fitsofitso yɛ shishigbɛ. Kɛfata QA kɛ QB quinones lɛ he lɛ, ajara chelated UQ molecules enyɔ (ni yɔɔ RC kɛ LH1 ŋwɛibɔfoi lɛ ateŋ) yɛ RC-LH114-W gbɛjianɔtoo lɛ mli yɛ amɛyitso kui ni atsu he nii jogbaŋŋ lɛ anaa (ni yɔɔ Q1 kɛ Q2, yɛ amɛteŋ). ŋwɛiniiaŋ). Mfoniri 5C). Akɛ isoprene henɔi enyɔ ha Q1, ni density shikpɔŋ he mfoniri lɛ tsuɔ isoprene 10 ni yɔɔ Q2 lɛ fɛɛ he nii. Yɛ RC-LH116 lɛ gbɛjianɔtoo lɛ mli lɛ, atsu chelated UQ10 molecules etɛ (Q1 kɛyashi Q3, Mfoniri 5D) ahe nii, ni molecules lɛ fɛɛ yɛ density ni yɔɔ faŋŋ yɛ tsitsi lɛ mli fɛɛ (Mfoniri 5, D kɛyashi G). Yɛ gbɛjianɔtoi enyɔ lɛ amli lɛ, quinone yitso kui ni ji Q1 kɛ Q2 lɛ agbɛhei lɛ yɛ tsakpaa ni hi jogbaŋŋ (Mfoniri S12F), ni amɛkɛ RC pɛ tsuɔ nii. Q1 lɛ yɛ RC-LH114-W lɛ W gap lɛ shinaa naa (Mfoniri 1G kɛ 5, C, D kɛ E), ni Q2 lɛ hu bɛŋkɛ he ni QB lɛ fiɔ he lɛ (Mfoniri 5, C, D) kɛ F). L-Trp143 kɛ L-Trp269 nibii ni eshwɛ ni ato he gbɛjianɔ lɛ bɛŋkɛ Q1 kɛ Q2 waa ni amɛhaa π-stacking nifeemɔi ni baanyɛ aba (Mfoniri 5, E kɛ F, kɛ Mfoniri S12). L-Gln88, 3.0 Å kɛjɛ Q1 kɔɔyɔɔ ni yɔɔ shɔŋŋ lɛ mli lɛ haa hydrogen ni mli wa (Mfoniri 5E); nɛkɛ sɛɛkuu nɛɛ ji nɔ ni ato he gbɛjianɔ yɛ RCs lɛ fɛɛ amli ja wekukpaa ni yɔɔ shɔŋŋ fe fɛɛ lɛ pɛ (Mfoniri S13). L-Ser91 ji nɔ ni akɛtoɔ Thr najiaŋ yɛ RCs krokomɛi babaoo mli (Mfoniri S13), eji 3.8 Angstroms kɛjɛ methyl oxygen ni yɔɔ Q1 lɛ mli, ni ebaanyɛ eha hydrogen ni bɛ hewalɛ (Mfoniri 5E). Etamɔ nɔ ni Q3 lɛ bɛ tsakpaa pɔtɛɛ ko, shi eyɛ hydrophobic kpokpaa ni yɔɔ RC-M subunit lɛ kɛ LH1-α subunit 5 kɛyashi 6 teŋ lɛ nɔ (Mfoniri 5, D kɛ G). Q1, Q2 kɛ Q3 loo chelated quinones ni bɛŋkɛ lɛ hu etsu he nii yɛ Tch mli. Blɛoo, Trv. 970 kɛ Blc. Iris shishitoo mla (9, 10, 12) tsɔɔ yelikɛbuamɔ quinone ni akɛfiɔ he ko ni ato he gbɛjianɔ yɛ RC-LH1 complex lɛ mli (Mfoniri S12G). RC-LH114-W lɛ mli UQs enumɔ ni efite lɛ kɛ 5.8±0.7 ni yɔɔ UQs fɛɛ complex mli lɛ kpãa gbee jogbaŋŋ, yɛ be mli ni UQs etɛ ni efite ni yɔɔ RC-LH114-W lɛ mli lɛ baa shi fe The measured value of 6.2±0.3 areless are S4 (Fig. straktsa.
L kɛ M polypeptides ni ji pseudo-symmetric lɛ eko fɛɛ eko hiɛ TMH enumɔ ni amɛfeɔ heterodimer ni kɛ BChl dimer kome, BChl monomers enyɔ, bacteriophage (BPh) monomers enyɔ, kɛ dade kome ni jeee Heme kɛ UQ10 molecules kome loo enyɔ feɔ ekome. Kɛtsɔ hydrogen ni yɔɔ ketone kuu lɛ naagbee lɛ nɔ kɛ bɔ ni ale akɛ ebua ehe naa yɛ Rps lɛ nɔ lɛ, akɛ carotenoids woɔ M-subunit lɛ mli, ni atsɛɔ lɛ cis-3,4-dehydroorhodopin. Henɔi (25). RC-H lɛ kponɔgbɛ fã lɛ ji nɔ ni TMH kome pɛ fiɔ fã lɛ he. RC lɛ he gbɛjianɔtoo muu lɛ fɛɛ tamɔ RC ni ji subunit etɛ ni ji bɔɔ nii ni yɔɔ tsakpaa lɛ (tamɔ Rba). ŋwɛibɔfoi (PDB ID: 3I4D). BChl kɛ BPh ni ji macrocycles lɛ, carotenoid sɛɛkuu lɛ kɛ dade ni jeee heme lɛ feɔ ekome yɛ nɛkɛ gbɛjianɔtoi nɛɛ amli yiŋkpɛɛ mli, taakɛ UQ10 yitso kuu ni yɔɔ QA he lɛ kɛ QB quinone ni yɔɔ RC-LH116 (Mfoniri S15) lɛ feɔ lɛ.
RC shishitoo mlai enyɔ ni yɔɔ QB hei srɔtoi ni yɔɔ lɛ haa hegbɛ hee ni akɛpɛiɔ tsakemɔi ni yaa nɔ ni fataa QB quinone ni akɛfiɔ he lɛ mli. Yɛ RC-LH116 tsɔne lɛ mli lɛ, QB quinone lɛ yɛ “he ni bɛŋkɛ” ni akɛfiɔ he kwraa lɛ mli (26), shi RC-LH114-W mligbalamɔ lɛ bɛ QB quinone. RC-LH114-W lɛ mli lɛ, QB quinone ko bɛ, ni enɛ haa mɔ naa kpɛɔ ehe ejaakɛ tsɔne lɛ tsuɔ nii, fe RC-LH116 tsɔne ni yɔɔ QB quinone ni ato he gbɛjianɔ jogbaŋŋ lɛ. Eyɛ mli akɛ LH1 ŋmɛlɛtswai enyɔ lɛ feɔ nɔ ni miihe ashɛ quinones enumɔ moŋ, shi enumɔ yɛ RC-LH116 ŋmɛlɛtswaa ni agbele lɛ mli, yɛ be mli ni etɛ pɛ ji nɔ ni ato he gbɛjianɔ yɛ RC-LH114-W ŋmɛlɛtswaa ni agbele lɛ mli. Nɛkɛ shishitoo mla ni yaa hiɛ nɛɛ baanyɛ ajie RC-LH114-W QB hei lɛ atsakemɔ oya, quinone kinetics ni yaa hiɛ oya yɛ tsɔne ni mli wa lɛ mli, kɛ bɔ ni ebaanyɛ efo LH1 loop lɛ mli lɛ kpo. 114-W lɛ mli UQ ni bɛ lɛ baanyɛ afee nɔ ni jɛ RC-LH114-W lɛ mli QB he lɛ etsɔ fɛ̃i amrɔ nɔŋŋ yɛ UQ sɛɛkuu mli The specific stage (akɛ QB he lɛ shinaa lɛ efo nɛkɛ conformation lɛ mli) ref.
Kɛji akɛ QB bɛ lɛ, L-Phe217 ni fata he lɛ sɛɛtsimɔ kɛyaa he ko ni kɛ UQ10 ni akɛfiɔ he lɛ kpãaa gbee, ejaakɛ ebaaha ekɛ klɛŋklɛŋ isoprene unit ni yɔɔ tsitsi lɛ mli lɛ akpe yɛ he ko (Mfoniri 6A). Kɛfata he lɛ, tsakemɔi titri ni yɔɔ faŋŋ ni kɔɔ bɔ ni shihilɛ lɛ yɔɔ ha lɛ he lɛ yɛ faŋŋ, titri lɛ, helix de (helix kuku ni yɔɔ loop ni yɔɔ TMH D kɛ E teŋ lɛ mli) he ni L-Phe217 lɛ jieɔ kɛyaa QB ni akɛfiɔ kotoku lɛ he lɛ kɛ L-Tyr223 lɛ sɛɛtsimɔ (Mfoniri 6A ) Koni akumɔ hydrogen ni akɛfiɔ he lɛ kɛ QB binding framework lɛ kɛ M- he ni eyɔɔ lɛ (Mfoniri 6B). Helix de pivots yɛ eshishigbɛ, Cα ni ji L-Ser209 lɛ tsiɔ ehe kɛ 0.33Å, yɛ be mli ni L-Val221Cα lɛ tsiɔ ehe kɛ 3.52Å. Tsakemɔi komɛi bɛ ni anaa yɛ TMH D kɛ E mli, ni ji nɔ ni akɛwoɔ nɔ yɛ shishitoo mlai enyɔ lɛ fɛɛ mli (Mfoniri 6A). Bɔ ni wɔle lɛ, enɛ ji klɛŋklɛŋ gbɛjianɔtoo ni yɔɔ adebɔɔ mli RC ni tsiɔ QB he lɛ naa lɛ. Kɛ akɛto shishitoo mla ni yeɔ emuu (QB-bound) lɛ he lɛ, etsɔɔ akɛ dani quinone lɛ baa shi lɛ, ehe miihia ni afee tsakemɔ ko yɛ bɔ ni eyɔɔ lɛ mli koni ebote quinone lɛ mli. L-Phe217 lɛ tsɔɔ ehe koni ekɛ quinone yitso kuu lɛ afee π-stacking nifeemɔ, ni helix lɛ shiɔ kɛyaa kponɔgbɛ, ni ehaa L-Gly222 fãji lɛ kɛ L-Tyr223 lɛ sɛɛ fã lɛ feɔ hydrogen tsakpaa ni yɔɔ hydrogen tsakpaa ni yɔɔ shiŋŋ (Mfoniri 6, A kɛ C).
(A) Hologram (L ŋwɛi ŋta, ŋmei/M ŋta, magenta) kɛ apo (ŋmei) he mfoniri ni feɔ ekome, ni ajieɔ nibii ni eshwɛ ni he hiaa waa lɛ akpo yɛ gbɛ ni tamɔ tso nɔ. UQ10 lɛ damɔ shi kɛha ŋmei ŋmɔŋ. Line ni yɔɔ ŋwɛigbɛ lɛ tsɔɔ hydrogen ni feɔ ekome yɛ ŋwɛibɔfoi lɛ fɛɛ amli lɛ. (B kɛ C) Apolipoprotein lɛ hiɛ he mfoniri lɛ kɛ ŋwɛi ŋta lɛ fɛɛ, ni maa L-Phe217 lɛ sɛɛ ŋwɛi ŋta ni ji oxygen lɛ nɔ mi yɛ bluu mli kɛ L-Tyr223 ni yɔɔ etsuru mli lɛ nɔ mi. L subunit lɛ ji ŋmɔtɔ; M kɛ H subunits lɛ bɛ kɔla. (D kɛ E) Apolipoprotein (D) kɛ muu lɛ fɛɛ (E) RC QB hei [su kɛtsɔ (A) nɔ] kɛ Thermophilus thermophilus PSII (eŋɔli, bluu kɛ plastic quinone; PDB ID: 3WU2) Akɛto (58).
Yɛ gbɛ ni akpaaa gbɛ nɔ lɛ, eyɛ mli akɛ RCs ni bɛ QB ni LH1 bɛ mli lɛ ahe gbɛjianɔtoi babaoo yɛ moŋ, shi abɔko tsakemɔi ni ana yɛ nikasemɔ nɛɛ mli lɛ he amaniɛ tsutsu. Nɔ ni fata enɛɛmɛi ahe ji QB sɛɛkuu gbɛjianɔtoo ni jɛ Blc. (PDB ID: 3PRC) (27), Tch. (28) kɛ Rba. sphaeroides (PDB ID: 1OGV) (29), ni fɛɛ miihe afee ekome kɛ amɛ QB gbɛjianɔtoo muu lɛ fɛɛ. 3PRC lɛ mlikwɛmɔ ni bɛŋkɛ kpaakpa lɛ jie lɛ kpo akɛ LDAO (Lauryl Dimethyl Amine Oxide) ni ji tsofa ni akɛjieɔ muji lɛ mli nibii lɛ fĩɔ amɛhe yɛ QB gbɛhe lɛ shinaa naa, ni baanyɛ atsĩ gbɛjianɔtoo ekoŋŋ ni efee nɔ ko ni agbe naa lɛ naa. Eyɛ mli akɛ LDAO lɛ fiteee yɛ he kome yɛ 1EYS loo 1OGV mli moŋ, shi akɛ tsofa kome too lɛ nɔŋŋ tsuɔ nii kɛsaa RC nɛɛ, ni no hewɔ lɛ ebaanyɛ ekɛ nɔ ni tamɔ nakai nɔŋŋ aba. Rba kristalo lɛ he gbɛjianɔtoo lɛ. Etamɔ nɔ ni Sphaeroides RC ni akɛ cytochrome c2 (PDB ID: 1L9B) fee ekome lɛ hu yɛ QB he ko ni agbe naa. Shi, yɛ sane nɛɛ mli lɛ, N-terminal kpokpaa ni yɔɔ RC-M polypeptide lɛ nɔ lɛ (ni kɛ QB ni akɛfiɔ he lɛ tsuɔ nii kɛtsɔɔ H ni akɛfiɔ Tyr ni eshwɛ yɛ Q helix lɛ nɔ lɛ nɔ) lɛ heɔ gbɛjianɔtoo ko ni jeee adebɔɔ nɔ, ni akwɛɛɛ QB tsakemɔ lɛ mli jogbaŋŋ (30 ). Nɔ ni haa mɔ tsui nyɔɔ emli ji akɛ, wɔnako nɛkɛ M polypeptide lɛ mli tsakemɔ ni tamɔ nɛkɛ yɛ RC-LH114-W shishitoo lɛ mli, ni miihe ashɛ nɔ kome kɛ N-terminal kpokpaa ni yɔɔ RC-LH116 RC lɛ nɔ lɛ. Esa akɛ wɔkadi hu akɛ, beni ajie LH1 antenna ni akɛ detergent tsuɔ nii yɛ mli lɛ sɛɛ lɛ, atsu apolipoprotein RCs ni yɔɔ PDB lɛ mli lɛ ahe nii, ni no jie quinone pools kɛ lipids ni yɔɔ RC lɛ kɛ LH1 ring ni ebɔle ehe lɛ mligbɛ ŋwɛigbɛ lɛ teŋ lɛ (31, 32). RC lɛ yaa nɔ etsuɔ ​​nii ejaakɛ ehiɛɔ cofactors lɛ fɛɛ mli, ja QB quinone ni fiteɔ lɛ pɛ, ni ehiɛɛɛ shi jogbaŋŋ ni bei pii lɛ elaajeɔ yɛ hesaamɔ be lɛ mli (33). Kɛfata he lɛ, ale akɛ LH1 kɛ adebɔɔ mli cyclic lipids ni ajieɔ kɛjɛɔ RC mli lɛ baanyɛ aná nitsumɔi anɔ hewalɛ, tamɔ charge-separated P+QB-state lɛ wala be ni afee kuku (31, 34, 35). No hewɔ lɛ, wɔsusuɔ akɛ, LH1 ŋwɛi ŋta ni bɔle RC lɛ he lɛ ni yɔɔ lɛ baanyɛ aha QB he ni “agbe naa” lɛ ahi shi, ni no baaha akutso lɛ mli shihilɛ ni bɛŋkɛ QB lɛ ahi shi.
Eyɛ mli akɛ apolipoprotein (ni QB quinone bɛ mli) kɛ gbɛjianɔtoo muu lɛ fɛɛ damɔ shi kɛha QB he lɛ tsakemɔ lɛ he mfonirii enyɔ pɛ moŋ, shi jeee nibii ni tee nɔ lɛ ateŋ nɔ ni nyiɛ sɛɛ lɛ, shi nibii komɛi yɛ ni tsɔɔ akɛ abaanyɛ atswa tsɔne ni akɛfiɔ he lɛ naa koni akɛtsi hydroquinone lɛ naa koni ekɛtsi substrate lɛ naa. Nɔ ni quinolol kɛ quinone ni bɛŋkɛ QB he ni apolipoprotein yɔɔ lɛ baanyɛ afee srɔto, ni no haa RC kpooɔ lɛ. Akɛ be babaoo ewie akɛ tsakemɔi ni baa yɛ bɔ ni nibii yɔɔ ha lɛ mli lɛ tsuɔ gbɛfaŋnɔ ko he nii yɛ quinones ni akɛfiɔ he kɛ nɔ ni baa shi lɛ mli. RCs ni edɔ lɛ ahewalɛ ni amɛkɛbaa quinones shi yɛ duŋ tsakemɔ sɛɛ lɛ efiteɔ (36); X-ray crystallography tsɔɔ akɛ nɛkɛ fitemɔ nɛɛ jɛ QB quinones ni amɔmɔ amɛ yɛ “sɛɛgbɛ” shihilɛ ko mli aaafee 4.5 Å kɛjɛ he ni bɛŋkɛ ni tsuɔ nii lɛ he (26) , 37). Wɔkɛɔ akɛ nɛkɛ distal binding conformation nɛɛ ji mfoniri ni tsɔɔ shihilɛ ni yɔɔ apolipoprotein kɛ ring structure muu lɛ fɛɛ teŋ lɛ mli, ni nyiɛɔ shishijee nifeemɔ ni kɛ quinone kɛ QB he lɛ gbelemɔ sɛɛ.
RC ni ji II ni anaa yɛ PSII complex ni ji fototrofik bacteria komɛi kɛ cyanobacteria, algae kɛ tsei lɛ yɛ shishitoo kɛ nitsumɔ he kwɛmɔ (38). (D kɛ E) lɛ maa bɔ ni PSII RCs lɛ kɛ QB he ni bacteria RC complex lɛ yɔɔ lɛ yeɔ egbɔ lɛ nɔ mi. Nɛkɛ nɔtomɔ nɛɛ efee nɔkwɛmɔnɔ be kakadaŋŋ kɛha gbɛjianɔtoi ni bɛŋkɛ kpaakpa ni kɔɔ quinone ni akɛfiɔ he kɛ nɔ ni baa shi lɛ he nikasemɔ. Woji ni eho lɛ tsɔɔ akɛ tsakemɔi ni baa yɛ bɔ ni mɔ ko yɔɔ ha lɛ mli lɛ fataa PSII ni baa quinones lɛ nɔ lɛ he (39, 40). No hewɔ lɛ, kɛji akɛ asusu RC lɛ sutsakemɔ mli nibii ni ato he gbɛjianɔ lɛ he lɛ, nɛkɛ gbɛ ni akɛfiɔ nii ahe ni anaaa tsutsu nɛɛ baanyɛ atsu nii yɛ QB he ni PSII RC lɛ yɔɔ lɛ hu yɛ tsei ni haa kɔɔyɔɔ ni haa wala yibii lɛ yeɔ nii lɛ amli.
Rps ΔpufW (pufW ni ajieɔ kɛjɛɔ mli ni akadiko nɔ) kɛ PufW-His (C-naagbee 10x E-kadi protein-W ni ajieɔ lɛ kpo kɛjɛɔ adebɔɔ mli pufW he lɛ) henɔi. palustris CGA009 lɛ ji nɔ ni atsɔɔ mli yɛ wɔ nitsumɔ ni eho lɛ mli (16). Ajie nɛkɛ tsɛŋemɔ helai nɛɛ kɛ fɔlɔ ni ji isogenic koo mli nɔ lɛ kɛjɛ friza lɛ mli kɛtsɔ tsɛŋemɔ helai fioo komɛi ni akɛwoɔ PYE (eko fɛɛ eko 5 g liter -1) (ni akɛto LB mli yɛ -80 °C, ni 50% ( w/v) glycerol) protein, yibii ni ajieɔ kɛjɛɔ mli kɛ succinate (%) agar/w yɔɔ mli lɛ nɔ. Akɛ plɛte lɛ wo duŋ mli nyɔɔŋ kome yɛ tsu mli latsaa mli yɛ shihilɛi ni kɔɔyɔɔ bɛ mli lɛ shishi, ni no sɛɛ lɛ akɛ kane yɛŋ (~50 μmolm-2 s-1) ni OSRAM 116-W halogen bulbs (RS Components, UK) kɛha lɛ ha lɛ mli gbii 3 kɛyashi 5 kɛyashi tsɛŋemɔ hela kome je kpo. Akɛ ŋmɔ kome tsu nii ni akɛwo M22+ mli 10 ml (41) ni akɛ 0.1% (w/v) casamino acids (ni sɛɛ mli lɛ atsɛɔ lɛ M22) fata he lɛ mli. Akɛ ŋmɔshi nibii lɛ wo shihilɛ ni kɔɔyɔɔ ni haa wala yibii lɛ faaa yɛ mli lɛ shishi yɛ duŋ mli yɛ 34°C kɛ 180 rpm ni hosoɔ ŋmɛlɛtswai 48, ni no sɛɛ lɛ, awo ŋmɔshi nibii lɛ mli 70 ml yɛ shihilɛi ni tamɔ nakai nɔŋŋ mli ŋmɛlɛtswai 24. Akɛ semi-aerobic culture ni falɛ ji 1 ml tsuɔ nii koni akɛwo M22 mli 30 ml mli yɛ 30 ml universal screw-top glase tsinsii ni mli tse mli ni akɛ la ni haa mɔ tsui nyɔɔ emli (~50μmolm-2 s-1) shwieɔ nɔ ŋmɛlɛtswai 48 kɛtsɔ sterile magnetic force Stirring rod nɔ. No sɛɛ lɛ, akɛ 30 ml ni ji culture lɛ wo culture aaafee litre 1 mli yɛ shihilɛi ni tamɔ nakai nɔŋŋ mli, ni no sɛɛ lɛ akɛwo culture aaafee litre 9 ni kpɛɔ yɛ ~200 μmolm-2 s-1 mli lɛ mli ŋmɛlɛtswai 72. Akɛ 7132 RCF lɛ mli minitii 30 tsu nii, ni akɛwo ~10 ml ni ji 20 mM tris-HCl (pH 8.0) mli ekoŋŋ, ni akɛto -20°C kɛyashi ehe baahia.
Kɛji akɛ ŋmɔtɔ lɛ shwie shi lɛ, okɛ kristalo komɛi ni ji deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) kɛ Roche holoenzyme protease inhibitor tsofai enyɔ (Merck, UK) afata wala yibii ni ajie lɛ ekoŋŋ lɛ ahe. Yɛ French tsɔne ko ni tsiimɔ ji 20,000 psi (Aminco, U.S.A.) mli lɛ, atsake tsɔnei lɛ amli shii 8 kɛyashi 12. Beni ajie wala yibii ni ekumɔko kɛ sũ ni nyɛɛɛ atsɔ mli lɛ kɛtsɔ 18,500 RCF nɔ minitii 15 yɛ 4°C nɔ lɛ, ajie membrane lɛ kɛjɛ lysate ni yɔɔ ŋmɔlɔŋmɔlɔ lɛ mli kɛtsɔ 113,000 RCF nɔ ŋmɛlɛtswai 2 yɛ 43,000°C nɔ. 100 kɛyashi 200 ml ni ji 20 mM tris-HCl (pH 8.0) lɛ mli ni okɛfee nɔ kome kɛyashi be mli ni nɔ ko ni anaa lɛ bɛ. Akɛ tsɔne ni akɛfoɔ tsɔne lɛ mli lɛ wo 20 mM tris-HCl (pH 8.0) (Anatrace, USA) ni 2% (w/v) β-DDM yɔɔ mli lɛ mli ŋmɛlɛtswaa kome yɛ duŋ mli yɛ 4°C kɛ tsɔne ni akɛshwieɔ shi blɛoo. No sɛɛ lɛ, okɛ centrifuge awo 70°C mli koni ekɛ RCF 150,000 lɛ atsake yɛ 4°C mli ŋmɛlɛtswaa kome koni ejie nibii ni eshwɛ ni nyɛɛɛ atsɔ mli lɛ.
Akɛ 50 ml DEAE Sepharose ion tsakemɔ ŋtayaa ni ŋtayaai etɛ (CV) ni akɛwoɔ mli lɛ tsu nii [20 mM tris-HCl (pH 8.0) ni 0.03% (w / v) β-DDM yɔɔ mli]. Kɛ CV binding buffers enyɔ afɔ ŋwɛitsu lɛ he, ni no sɛɛ lɛ okɛ binding buffers enyɔ ni 50 mM NaCl yɔɔ mli lɛ afɔ ŋwɛitsu lɛ he. RC-LH116 tsɔne lɛ ji linear gradient ni ji 150 kɛyashi 300 mM NaCl (yɛ tsɔne ni akɛfiɔ nii ahe) yɛ 1.75 CV nɔ, ni tsɔne ni akɛfiɔ nii ahe ni eshwɛ lɛ ji nɔ ni akɛ tsɔne ni akɛfiɔ nii ahe ni 300 mM NaCl yɔɔ mli lɛ tsu nii yɛ 0.5 CV nɔ. 250 kɛ 1000 nm teŋ lɛ, ni okɛ fã ni yɔɔ ŋwɛi ŋta (A880/A280) ni fe 1 lɛ ahi 880 kɛyashi 280 nm, ni okɛwo mli shii enyɔ yɛ binding buffer lɛ mli, ni okɛ gbɛ kome too lɛ nɔŋŋ atsu nii ekoŋŋ yɛ DEAE column lɛ nɔ On purification. Dilute fãi ni A880/A280 ratios fe 1.7 kɛ A880/A805 ratios fe 3.0, feemɔ ion tsakemɔ fã ni ji etɛ lɛ, ni ohiɛ fãi ni A880/A280 ratios fe 2.2 kɛ A880/A805 ratios fe 0.0. 100,000 molecular weight cut-off (MWCO) centrifugal filta (Merck, UK), ni akɛwo Superdex 200 16/600 dalɛ ni ajieɔ kɛjɛɔ mli (GE Healthcare, US) ni buffer 200 m, 1.5 CV. 100 A880 kɛ A880/A280 ratios ni fe 2.4 kɛ A880/A805 ratios ni fe 5.8 kɛyashi 100 A880, ni okɛtsu nii amrɔ nɔŋŋ kɛha cryo-TEM grid hesaamɔ loo nitoo Keep at -8.
Akɛ tsɔne ni haa mɔ he jɔɔ lɛ ni jɛ PufW-His lɛ mli lɛ wo 20 ml HisPrep FF Ni-NTA Sepharose ŋwɛi ŋta (20 mM tris-HCl (pH 8.0) ni 200 mM NaCl kɛ 0.03% (w/w) yɔɔ mli lɛ mli) yɛ IMAC buffer ( GE) mli. v) β-DDM]. Akɛ IMAC buffer CV enumɔ tsuɔ nii kɛfɔɔ ŋwɛitsu lɛ he, ni no sɛɛ lɛ akɛ IMAC buffer CV enumɔ ni histidine 10 mM yɔɔ mli lɛ tsu nii. Akɛ IMAC buffers enumɔ ni histidine 100 mM yɔɔ mli lɛ jie shishijee nibii lɛ kɛjɛ ŋwɛitsu lɛ mli. 100,000 MWCO filter (Merck, UK), ni akɛ buffer ni fiɔ nii amli lɛ ewo mli shii 20, ni no sɛɛ lɛ akɛfataa 25 ml he yɛ DEAE b buffer lɛ mli kɛyaa. kɛtsɔ hiɛ. 100 mM NaCl (yɛ tsɔne ni akɛfiɔ nii ahe lɛ mli), ni CV ejwɛ ni eshwɛ lɛ ni 100 mM ni akɛfiɔ nii ahe lɛ yɔɔ mli lɛ, tsɔnei ni eshwɛ ni akɛfiɔ nii ahe lɛ chlobin kɛ so8de2 lɛ nɔ. 100,000 MWCO centrifugal filter mli, ni akɛ 1.5 CV IMAC eyi mli obɔ kɛtsɔ hiɛ Buffer equilibrated Superdex 200 ni akɛwo mli lɛ mli, ni akɛwo mli. 1.5 CV. Buamɔ ŋwɛi ŋta ni ji ŋwɛi ŋta lɛ mli lɛ naa ni okɛ A880/A280 ratios ni fe 2.1 kɛ A880/A805 ratios ni fe 4.6 kɛyashi 100 A880, ni akɛtsuɔ nii amrɔ nɔŋŋ kɛha fɛ̃i TEM grid he saamɔ loo akɛtoɔ -80°C he.
Akɛ Leica EM GP friza ni akɛwoɔ nu mli lɛ tsu nii ni akɛsaa TEM grids ni mli edɔɔ waa lɛ. Akɛ IMAC buffer lɛ wo A880 ni ji 50 mli, ni no sɛɛ lɛ akɛ 5μl wo akɔɔble tsɔne hee ko ni akɛ la ni tsoɔ waa ni ji QUANTIFOIL 1.2/1.3 carbon ni akɛwo mli lɛ nɔ (Agar Scientific, UK). Akɛ grid lɛ awo 20°C kɛ 60% relative humidity mli kɛyashi 30 s, no sɛɛ lɛ, ashɛre lɛ 3 s, ni no sɛɛ lɛ, agbe lɛ yɛ nu ni ji ethane mli yɛ -176°C.
Akɛ Titan Krios nibii bibii ni akɛkwɛɔ nibii bibii amli lɛ wo eBIC (Elektronik Bioimaging Center) (British Diamond La Jɛɛhe) lɛ nɔ, ni tsuɔ nii yɛ hewalɛ ni yaa oya ni ji 300kV, kɛ hewalɛ ni ji 130,000 ni ji eV × kɛ 130,000 ni ji Cho. Akɛ Gatan 968 GIF Quantum ni K2 ŋwɛi ŋta ni anaa lɛ tsu nii ni akɛŋmala mfoniri yɛ yibɔ ni akɛkaneɔ nii lɛ mli koni akɛbua saji anaa. Pixel dalɛ ni ato lɛ ji 1.048Å, ni tsofa lɛ falɛ ji 3.83 e-Å-2s-1. Ebua sini lɛ naa yɛ sɛkɛndii 11 mli ni ejara mli ewo fãi 40 mli. Kɛ he ni akɛ carbon ewo mli lɛ atsu nii ni okɛkwɛ nibii bibii lɛ anɔ ekoŋŋ, ni no sɛɛ lɛ, obua sinii etɛ anaa yɛ bu fɛɛ bu mli. Yɛ fɛɛ mli lɛ, abua sinii 3130 naa, ni defocus values ​​lɛ yɛ -1 kɛ -3μm teŋ.
Akɛ nibii bibii ni akɛkwɛɔ nibii bibii ni yɔɔ Asterbury Biostructure Laboratory (Leeds Yunivɛsiti, UK) lɛ nɔŋŋ tsu nii kɛbua saji ni kɔɔ RC-LH116 he lɛ anaa. Abua saji lɛ anaa yɛ kanemɔ gbɛ nɔ kɛ 130 k, ni akɛ pixel dalɛ to 1.065 Å kɛ 4.6 e-Å-2s-1. Akɛ sɛkɛndii 12 wo sini lɛ mli, ni aja mli awo fãi 48 mli. Yɛ fɛɛ mli lɛ, abua sinii 3359 naa, ni defocus values ​​lɛ yɛ -1 kɛ -3μm teŋ.
Atsuɔ saji fɛɛ ahe nii yɛ Relion 3.0 paipii lɛ mli (42). Kɛ Motioncorr 2 (43) atsu nii ni okɛsaa ŋwɛi ŋta lɛ nyiɛmɔ kɛtsɔ tsofa ni akɛwoɔ mli lɛ nɔ, ni no sɛɛ lɛ okɛ CTFFIND 4.1 (44) atsu nii ni okɛtsɔɔ CTF (srɔtofeemɔ nitsumɔ) paramita lɛ. Atsɔɔ mfoniri bibii ni ajieɔ yɛ shishijee nitsumɔ nɛɛ sɛɛ yɛ mfoniri 2. S16. Akɛ otomatik halamɔ tɛmplate lɛ feɔ kɛtsɔ nine ni akɛhalaa aaafee 250 pixels ni ji 1000 mli nibii bibii ni yɔɔ 250-pixel frem mli ni nɔ ko bɛ ni tsɔɔ nɔ ni ji enyɔ (2D) mligbalamɔ, ni no haa akpooɔ nakai mligbalamɔi ni kɛ nɔkwɛmɔnɔ mujiwoo kpeɔ loo bɛ sui ni anaa lɛ. No sɛɛ lɛ, afee otomatik halamɔ yɛ mfoniri bibii lɛ fɛɛ anɔ, ni RC-LH114-W lɛ ji nibii bibii 849,359, ni RC-LH116 complex lɛ ji nibii bibii 476,547. Nibii ni ahala lɛ fɛɛ etsɔ 2D mligbalamɔ ni jeee nɔ ni akɛtsɔɔ mli lɛ mli shii enyɔ, ni yɛ foidamɔ fɛɛ foidamɔ sɛɛ lɛ, akpooɔ nibii bibii ni kɛ carbon kpokpaa lɛ kpeɔ lɛ, nɔkwɛmɔnɔ ni mli ewo muji, nibii ni yɔɔ faŋŋ ni bɛ loo nibii bibii ni feɔ ekome waa lɛ, ni nɔ ni jɛɔ mli baa ji 772,033 (90.9%) kɛ 359.578% of Parles3 (359,6578 ) ni akɛtsuɔ nii. RC-LH114-W kɛ RC-LH116 lɛ mli. Akɛ stochastic gradient shigbeemɔ gbɛ lɛ tsu nii kɛfee shishijee 3D nɔkwɛmɔnɔ lɛ. Kɛtsɔ shishijee nɔkwɛmɔnɔ lɛ nɔ akɛ nɔ ni akɛtaoɔ saji amli lɛ, ajaa nibii bibii ni ahala lɛ amli awoɔ kui ejwɛ mli yɛ 3D mli. Kɛtsɔ nɔkwɛmɔnɔ ni yɔɔ nɛkɛ kuu nɛɛ mli lɛ nɔ lɛ, feemɔ 3D refining yɛ nibii bibii ni yɔɔ kuu ni da fe fɛɛ lɛ mli lɛ anɔ, no sɛɛ lɛ okɛ shishijee 15Å low-pass filter lɛ atsu nii ni okɛha solvent he lɛ nɔ, okɛ 6 pixels ni yɔɔ blɛoo lɛ afata he, ni okɛ pixels lɛ atsu nii yɛ sɛɛ mli koni okɛsaa Gatan K2 transfer function ni ji ŋwɛi ŋta lɛ. Kɛha RC-LH114-W dataset lɛ, atsake shishijee nɔkwɛmɔnɔ nɛɛ kɛtsɔ ŋmɔlɔ ni mli wa ni yɔɔ mask lɛ naa lɛ ni ajieɔ lɛ nɔ (ni ajie kɛjɛ ŋmɔlɔ ni mli wa ni yɔɔ UCSF Chimera lɛ mli lɛ mli). Akɛ nɔkwɛmɔnii ni jɛ mli ba lɛ (RC-LH114-W kɛ RC-LH116 lɛ yiŋkpɛɛi lɛ ji 3.91 kɛ 4.16 Å, yɛ amɛteŋ) tsuɔ nii akɛ nɔ ni akɛtsɔɔ 3D mligbalamɔ ni ji enyɔ lɛ mli. Akɛ nibii bibii ni akɛtsuɔ nii lɛ toɔ shishijee 3D klas lɛ mli ni amɛbɛ tsakpaa ni mli wa kɛ akutso lɛ mli. Nɔ ni feɔ ekome loo nɔ ni yɔɔ faŋŋ ni bɛ. Yɛ 3D mligbalamɔ ni ji enyɔ lɛ sɛɛ lɛ, ahala kuu ni yɔɔ ŋmɛlɛtswaa ni nɔ kwɔ fe fɛɛ lɛ [Kɛha RC-LH114-W lɛ, kuu kome ji 377,703 mli nibii bibii (44.5%), kɛha RC-LH116 lɛ, kui enyɔ pɛ yɔɔ, ni amɛ fɛɛ amɛyibɔ ​​ji 260,752 mli nibii bibii (4%) the W. yɛ shishijee sɛɛkuu lɛ sɛɛ kɛ srɔtofeemɔ fioo ko]. Ajieɔ nibii bibii ni ahala lɛ ekoŋŋ yɛ adeka ko ni hiɛ pixel 400 mli, ni akɛ 3D refining tsuɔ he nii. Akɛ shishijee 15Å low-pass filta, 3 pixel shikpɔŋ he mfoniri mlilɛɛmɔ kɛ 3 pixel soft mask tsuɔ nii kɛfeɔ solvant mask lɛ. Akɛ CTF ni akɛtsuɔ nii yɛ fã fɛɛ fã mli lɛ tsuɔ nii, ni akɛtsakeɔ fã fɛɛ fã ni tsiɔ ehe lɛ kɛ CTF ni akɛtsuɔ nii yɛ fã kome mli lɛ mli fã ni ji enyɔ lɛ, 3D tsakemɔ, solvent masking kɛ post-processing tsuɔ nii yɛ gbɛ fɛɛ gbɛ nɔ koni akɛtsake nɔ ni jɛɔ mli baa lɛ mli jogbaŋŋ. Kɛ akɛ FSC (Fourier Shell Correlation Coefficient) ni ji 0.143 lɛ tsu nii lɛ, RC-LH114-W kɛ RC-LH116 naagbee nɔkwɛmɔnii lɛ ahe yiŋkpɛɛi lɛ ji 2.65 kɛ 2.80Å, yɛ amɛteŋ. Atsɔɔ FSC curve ni ji naagbee nɔkwɛmɔnɔ lɛ yɛ mfoniri 2. S17.
Akɛ protein ni nyiɛɔ sɛɛ lɛ fɛɛ jieɔ kɛjɛɔ 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). Akɛ SWISS-MODEL (45) tsu nii ni akɛfee RC he mfoniri ni tamɔ nɔ kome, ni RC-L, RC-M kɛ RC-H mli protein ni nyiɛɔ sɛɛ kɛ Rba kristalo shishitoo lɛ yɔɔ mli. RC lɛ ji nɔ ni akɛtsu nii akɛ nɔkwɛmɔnɔ (PDB ID: 5LSE) (46). Kɛ “shikpɔŋ he mfoniri ni sa” dɛŋdade ni yɔɔ UCSF Chimera lɛ mli lɛ atsu nii ni okɛ nɔkwɛmɔnɔ ni afee lɛ akpã shikpɔŋ he mfoniri lɛ he (47), ni oha protein lɛ he gbɛjianɔtoo lɛ aya hiɛ, ni okɛ cofactor [4×BChl a (monomer wojiatoohe lɛ gbɛi = BCL), 2×BPh a (BPH), UQ10 henɔ kome loo enyɔ (U10), ekome ni jeee he-heron . 3,4-dihaidrɔheksakarbonilkolin (QAK)] kɛ Coot (48) tsuɔ nii kɛfataa he. Akɛni QAK bɛ monomer wolotoohe lɛ hewɔ lɛ, akɛ eLBOW dɛŋdade ni yɔɔ PHENIX (49) lɛ tsu nii kɛfee parameter.
Nɔ ni nyiɛ sɛɛ ji, ama LH1 fã bibioo lɛ. Shishijee lɛ, akɛ otomatik tsumaa dɛŋdade ni yɔɔ PHENIX (49) lɛ mli lɛ tsu nii ni akɛma LH1 sɛɛkuu lɛ fã ko kɛtsɔ shikpɔŋ he mfoniri lɛ kɛ LH1-α kɛ LH1-β protein sɛɛkuu lɛ nɔ akɛ nɔ ni akɛwoɔ mli. Halamɔ LH1 subunit ni yeɔ emuu fe fɛɛ lɛ, jiemɔ ni okɛwo Coot mli, ni okɛ onine afata nɔ ni nyiɛ sɛɛ ni elaaje lɛ he, ni okɛ onine atsake gbɛjianɔtoo muu lɛ fɛɛ mli dani okɛ BCls enyɔ a (BCL) kɛ spirilloxanthin (CRT) afata he [tamɔ bɔ ni Rps ni kɔɔ he lɛ tsɔɔ lɛ LH1 complex lɛ mli tsɔne lɛ mli tsɔne lɛ mli tsɔne lɛ kɛ nɔ ni ale lɛ. Henɔi (17)]. Kpɔ LH1 fã bibioo lɛ fɛɛ he mfoniri, ni okɛ UCSF Chimera “Docking Map Tool” lɛ atsu nii ni okɛma he ni bɛŋkɛ ni jeee nɔkwɛmɔnɔ ni LH1 density lɛ yɔɔ lɛ, ni no sɛɛ lɛ okɛtsake lɛ yɛ Coot mli; sɛɛkuu nifeemɔ lɛ mli kɛyashi be mli ni afee LH1 subunits lɛ fɛɛ he nɔkwɛmɔnɔ. Kɛha RC-LH114-W gbɛjianɔtoo lɛ, kɛtsɔ tsɔne ni akɛhaaa lɛ ni ajieɔ yɛ Coot lɛ mli lɛ nɔ lɛ, ajieɔ protein lɛ kɛjɛɔ nibii ni eshwɛ ni jeee protein lɛ amli yɛ USCF Chimera shikpɔŋ he mfoniri lɛ mli ni akɛ Autobuild dɛŋdade lɛ tsuɔ nii kɛtoɔ shishijee nɔkwɛmɔnɔ lɛ shishi, kɛ subunits ni eshwɛ lɛ (protein-W) Modeling. Yɛ FINIS (49) lɛ mli. Kɛ nɔ ni elaaje lɛ afata nɔ ni jɛ mli ba lɛ he yɛ Coot (48) mli, ni no sɛɛ lɛ okɛ onine atsake fã bibioo lɛ fɛɛ. Nibii ni eshwɛ ni ajieee lɛ kpo lɛ kɛ lipids ni akɛfee ekome lɛ kpãa gbee (PDB monomer wolotoohe ID ni ji CDL = CDL, POPC = 6PL kɛ POPG = PGT), β-DDM nibii ni akɛjieɔ muji (LMT) kɛ UQ10 mli nibii (U10) lɛ. Kɛ PHENIX optimization (49) kɛ manual optimization yɛ Coot (48) mli lɛ atsu nii ni okɛha shishijee nɔkwɛmɔnɔ lɛ fɛɛ aye emuu kɛyashi nɔkwɛmɔnɔ lɛ he akɔntaabuu kɛ nɔ ni anaa lɛ mlihilɛ ni sa lɛ nyɛŋ aya hiɛ babaoo. Yɛ naagbee lɛ, kɛ LocScale (50) atsu nii ni okɛha maŋ lɛ shikpɔŋ he mfoniri lɛ afee mlɛo, ni no sɛɛ lɛ ofee sɛɛkuu krokomɛi babaoo ni akɛtsɔɔ bɔ ni mɛi ayibɔ ni akɛhaaa kɛ bɔ ni amɛ diɛŋtsɛ amɛfeɔ amɛnii amɛhaa lɛ mli.
Atsɔɔ peptides, cofactors kɛ lipids kɛ quinones krokomɛi ni yɔɔ amɛmli lɛ amli yɛ mfoniri 1 kɛ 2. S18 kɛyashi S23. Atsɔɔ naagbee nɔkwɛmɔnɔ lɛ he akɔntaabuu yɛ Table S1.
Kɛji akɛ atsɔɔɔ gbɛ kroko nɔ lɛ, abua UV/Vis/NIR hulutsoo be lɛ naa yɛ Cary60 hulutsoo tsɔne (Agilent, USA) nɔ yɛ 1 nm teŋ kɛjɛ 250 nm kɛyashi 1000 nm kɛ 0.1s ni akɛwoɔ mli lɛ mli.
Akɛ nɔkwɛmɔnɔ lɛ woɔ quartz cuvette ni 2 mm gbɛ kɛyaa A880 ni ji 1 mli lɛ mli, ni abua 400 kɛ 1000 nm teŋ ŋwɛi ŋtayaa lɛ naa. Abua ŋwɛi ŋtayaa ni yɔɔ ŋwɛigbɛ lɛ naa yɛ Jasco 810 ŋwɛi ŋtayaa nɔ (Jasco, Japan) yɛ 1 nm teŋ yɛ 400 nm kɛ 950 nm teŋ yɛ 20 nm min-1 mli.
Akɛ 10μl volume lɛ woɔ 990μl binding buffer loo methanol mli, ni abua ŋwɛi ŋtayaa lɛ naa amrɔ nɔŋŋ koni BChl fitemɔ aba shi. Akɛ BChl ni yɔɔ methanol nɔkwɛmɔnɔ fɛɛ nɔkwɛmɔnɔ mli lɛ to sɛɛfomɔ ni yɔɔ 771 nm ni ji 54.8 mM-1 cm-1 lɛ he, ni atsɔɔ sɛɛfomɔ ni yɔɔ mli lɛ (51). Jaa BChl ni asusu lɛ mli 32 (RC-LH114-W) loo 36 (RC-LH116) mli koni ona shishijee nibii ni yɔɔ mli lɛ, ni no sɛɛ lɛ akɛtsuɔ nii koni akɛna bɔ ni nɔkwɛmɔnɔ kome too lɛ nɔŋŋ ni abua naa yɛ buffer Extinction coefficient lɛ mli lɛ shwieɔ shi ehaa lɛ. kpaa gbɛ. Akɛ susumɔi etɛ ni afɔɔ feemɔ lɛ tsu nii kɛha nɔkwɛmɔnɔ fɛɛ nɔkwɛmɔnɔ, ni akɛ BChl Qy maximum lɛ mlijaa mlijaa lɛ tsu nii kɛbu he akɔntaa. RC-LH114-W ni sɛɛ foɔ ni asusuɔ yɛ 878 nm lɛ ji 3280±140 mM-1 cm-1, yɛ be mli ni RC-LH116 ni sɛɛ foɔ ni asusuɔ yɛ 880 nm lɛ ji 3800±30 mM-1 cm-1.
Akɛ UQ10 lɛ to gbɛ ni yɔɔ (52) lɛ naa. Yɛ kukufoo mli lɛ, akɛ Agilent 1200 HPLC gbɛjianɔtoo lɛ tsu nii kɛfee HPLC ni sɛɛ tsɛɔ (RP-HPLC) lɛ. 0.02 nmol ni ji RC-LH116 loo RC-LH114-W lɛ mli 50μl ni ji 50:50 methanol:chloroform ni 0.02% (w/v) ferric chloride yɔɔ mli lɛ mli, ni okɛ Beckman Coulter Ultrasphere O2 ni ato he gbɛjianɔ momo lɛ awo mli. 40°C yɛ HPLC tsofa (80:20 mɛtanɔl:2-propanol) mli yɛ ×25 cm ŋwɛitsu nɔ. 275 nm (UQ10), 450 nm (carotenoids) kɛ 780 nm (BChl) mli ŋmɛlɛtswaa kome. Akɛ 275 nm chromatogram lɛ mli nɔ ni fe fɛɛ lɛ fata he yɛ minitii 25.5 mli, ni nɔ kroko ni anyɛɔ ayɔseɔ lɛ bɛ mli. Akɛ he ni akɛfee ekome lɛ buɔ UQ10 ni ajie lɛ mli molar lɛ he akɔntaa kɛtsɔ calibration curve ni abu he akɔntaa kɛjɛ 0 kɛyashi 5.8 nmol (Mfoniri S14) ni akɛwoɔ mli lɛ mli lɛ nɔ. Akwɛ nɔkwɛmɔnɔ fɛɛ nɔkwɛmɔnɔ mli yɛ shii etɛ mli, ni tɔmɔ ni abɔ he amaniɛ lɛ kɛ SD ni yɔɔ mlijaa lɛ mli lɛ kpãa gbee.
Akɛ 30 μM ni ajie okpɔŋɔ tsui mli cytochrome c2 (Merck, UK) kɛ 0 kɛyashi 50 μMUQ2 (Merck, UK) lɛ fee tsofa ko ni RC-LH1 ni yɔɔ mli lɛ mli Qy ni fa fe fɛɛ ni ji 0.1 lɛ fee. Afee 1-ml nɔkwɛmɔnii etɛ yɛ UQ2 mli nibii ni yɔɔ mli lɛ eko fɛɛ eko mli ni akɛwo duŋ mli nyɔɔŋ kome yɛ 4°C koni ana akɛ etsake ehe kwraa kɛya duŋ lɛ mli dani asusu. Akɛ tsofa lɛ wo OLIS RSM1000 modular spectrophotometer ko ni akɛ 300 nm la/500 line ŋtayaa, 1.24 mm botemɔ mli, 0.12 mm teŋgbɛ kɛ 0.6 mm kpojee ŋtayaai ewo mli lɛ mli. Akɛ 600 nm long pass filta ko woɔ nɔkwɛmɔnɔ fototube lɛ shinaa naa kɛ tsɔɔmɔ fotomultiplier tube lɛ koni ajie la ni haa mɔ tsui nyɔɔ emli lɛ kɛjɛ mli. Akwɛ bɔ ni tsɔne lɛ shwieɔ shi ehaa lɛ nɔ yɛ 550 nm kɛ 0.15 s ni akɛwoɔ mli lɛ mli. Akɛ 880 nm M880F2 LED (Thorlabs Ltd., UK) lɛ tsuɔ nii kɛtsɔɔ fiber optic kpã nɔ yɛ 90% hewalɛ mli kɛtsɔɔ DC2200 nɔkwɛlɔ (Thorlabs Ltd., UK) nɔ, ni ajieɔ kɛyaa kane jɛɛhe ni ji°0 lɛ nɔ. Akɛ susumɔ la lɛ teɔ shi ewoɔ ashwishwɛ lɛ koni ekɛ la fɛɛ la ni shishijee lɛ nɔkwɛmɔnɔ lɛ ŋɔɔɔɔ lɛ aku sɛɛ aba. Kwɛmɔ absorbance lɛ nɔ 10 s dani la ni tsoɔ 50 s lɛ aba. No sɛɛ lɛ, akwɛ bɔ ni nu lɛ shwieɔ shi ehaa lɛ nɔ ekoŋŋ kɛyashi 60 s yɛ duŋ mli koni ana bɔ ni quinolol lɛ diɛŋtsɛ haa cytochrome c23 + lɛ baa shi eha (kwɛmɔ mfoniri S8 kɛha saji ni atsɔɔ mli lɛ).
Akɛ saji lɛ tsu nii kɛtsɔ linear initial rate ni akɛwoɔ 0.5 kɛyashi 10 s mli (ni damɔ UQ2 mlibotemɔ nɔ) kɛ average rates ni yɔɔ sample etɛ lɛ fɛɛ mli yɛ UQ2 mlibotemɔ fɛɛ mli lɛ nɔ. Akɛ RC-LH1 mlibotemɔ ni abu he akɔntaa kɛtsɔ sɛɛnamɔ ni jɛɔ mli baa lɛ nɔ lɛ tsu nii ni akɛtsake yibɔ lɛ awo nɔ ni haa mɔ tsui nyɔɔ emli lɛ mli, ni atsɔɔ yɛ Origin Pro 2019 (OriginLab, USA), ni akɛto Michaelis-Menten nɔkwɛmɔnɔ lɛ he koni akɛna Km kɛ Kcat sɛɛnamɔi ni yɔɔ faŋŋ lɛ.
Kɛha be kukuoo mli ŋmɔlɔ susumɔi lɛ, ajie RC-LH1 nɔkwɛmɔnɔ lɛ kɛyashɛ ~2μM yɛ IMAC buffer ni 50 mM sodium ascorbate (Merck, USA) kɛ 0.4 mM Terbutin (Merck, USA) yɔɔ mli lɛ mli. Akɛ Ascorbic acid tsuɔ nii akɛ afɔleshaa electron halɔ, ni akɛ tert-butaclofen tsuɔ nii akɛ QB inhibitor koni ekwɛ akɛ RC halɔ titri lɛ ahi shi akɛ nɔ ni baa shi (ni ji, ekɛ fotooxidized ehiɛɛɛ shi) yɛ susumɔ nifeemɔ lɛ mli fɛɛ. Akɛ nɔkwɛmɔnɔ ni miihe ashɛ 3 ml fataa kusum naa tsɔne ko ni tsiɔ ehe (aaafee 0.1 m yɛ elɛɛmɔ mli, 350 RPM) ni 2 mm optical gbɛ lɛ kɛlɛ ji lɛ he koni ana nɔmimaa akɛ nɔkwɛmɔnɔ ni yɔɔ laser gbɛ lɛ nɔ lɛ yɛ be ni fa bɔ ni sa kɛha duŋ mli tsakemɔ yɛ excitation pulses lɛ ateŋ. 100-fs laser pulses ni okɛha Ti: Sapphire laser gbɛjianɔtoo (Spectra Physics, USA) lɛ aya hiɛ ni okɛha nɔkwɛmɔnɔ lɛ mli ajɔ yɛ 880 nm yɛ 1 kHz (20 nJ kɛha NIR loo 100 nJ kɛha Vis) mli. Dani obaabua saji anaa lɛ, ha nɔkwɛmɔnɔ lɛ awo la ni haa mɔ tsui nyɔɔ emli lɛ mli aaafee minitii 30. Nibii ni ajieɔ lɛ kpo lɛ baaha QA lɛ akatsu nii (ekolɛ ebaaha QA lɛ aba shi shikome loo shii enyɔ). Shi ofainɛ kadimɔ akɛ nɛkɛ gbɛ nɛɛ baanyɛ aku sɛɛ aba ejaakɛ yɛ be kakadaŋŋ ni akɛtsakeɔ duŋ mli lɛ sɛɛ lɛ, RC baaku esɛɛ kɛya QA nitsumɔ lɛ mli fiofio. Akɛ Helios ŋwɛi ŋtayaa (Ultrafast Systems, USA) tsu nii kɛsusu ŋtayaai ni hoɔ oya kɛ be ni sɛɛ tsɛɔ ni ji -10 kɛyashi 7000 ps. Kɛ Surface Xplorer tsɔne (Ultrafast Systems, USA) atsu nii ni okɛjie saji ni ato lɛ, no sɛɛ lɛ okɛfee ekome ni okɛfee ekome. Kɛ CarpetView tsɔne ni akɛtsuɔ nii (Light Conversion Ltd., Lithuania) lɛ atsu nii ni okɛ saji ni akɛfee ekome lɛ atsu nii ni okɛna srɔtofeemɔ ni kɔɔ fitemɔ he, loo okɛ nitsumɔ ko ni kɔɔ ŋwɛibɔfoi babaoo ni yɔɔ dɛŋdade lɛ hetoo lɛ he lɛ atsu nii koni ekɛ ŋwɛibɔfoi ni yɔɔ Origin, USALa (Origin) lɛ mli lɛ akpã gbee.
Taakɛ atsĩ tã yɛ yiteŋgbɛ (53) lɛ, asaa fotosintɛtik sini ko ni LH1 complex yɔɔ mli ni RC kɛ peripheral LH2 antenna fɛɛ bɛ mli lɛ. Akɛ 20 mM tris (pH 8.0) wo mli, ni no sɛɛ lɛ, akɛwo quartz cuvette ko ni 2 mm ŋmɛlɛtswaa gbɛ tsɔɔ mli lɛ mli. Akɛ 30nJ laser pulse tsu nii koni akɛwo nɔkwɛmɔnɔ lɛ mli yɛ 540 nm kɛ sɛɛkuu be ni ji -10 kɛyashi 7000 ps. Tsɔɔmɔ data kuu lɛ he nii taakɛ atsɔɔ mli kɛha Rps. pal nɔkwɛmɔnɔ.
150,000 RCF nɔ ŋmɛlɛtswai 2 yɛ 4°C, ni no sɛɛ lɛ, atsake e-absorbance ni yɔɔ 880 nm lɛ awo 20 mM tris-HCl (pH 8.0) kɛ 200 mM NaCl mli. Akɛ 2% (w/v) β-DDM ni akɛwoɔ mli fiofio lɛ shwieɔ tsɔne lɛ mli ŋmɛlɛtswaa kome yɛ duŋ mli yɛ 4°C. 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) mli kɛyashɛ protein ni falɛ ji 2.5 mg ml-1 (Bio-Rad mlipɛimɔ) mli. Afee nitsumɔ kroko kɛjɛ gbɛ ni akala tsutsu lɛ nɔ (54), ni aje shishi kɛ 50 μg protein ni akɛwoɔ mli lɛ mli 50 μl TEAB ni 1% (w/v) sodium laurate (Merck, UK) yɔɔ mli lɛ fɛɛ. Yɛ sonication kɛha 60 s sɛɛ lɛ, ajie lɛ kɛ 5 mM tris (2-carboxyethyl) phosphine (Merck, UK) yɛ 37 ° C kɛha minitii 30. Kɛha S-alkylation lɛ, okɛ 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) awo nɔkwɛmɔnɔ lɛ mli ni okɛfata he kɛjɛ 200 mM isopropanol stɔk solution mli minitii 10 yɛ shĩa mli latsaa mli. Akɛ trypsin/endoproteinase Lys-C ni akɛfutuɔ nii (Promega UK) ni ji 2 μg fata he lɛ tsu nii, ni akɛwo 37°C mli ŋmɛlɛtswai 3. Akɛ 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) fata he, ni akɛwo mli kɛyashi 60 s. 15,700 RCF minitii 5 lɛ, atswa fã mligbalamɔ lɛ he hewalɛ. Taakɛ mɔ ni fee lɛ gbɛjianɔtoo lɛ tsɔɔ lɛ, akɛ C18 spin column (Thermo Fisher Scientific, UK) tsu nii ni akɛjie shishigbɛ fã ni peptide lɛ yɔɔ mli lɛ jogbaŋŋ ni ajie ŋmei kɛjɛ mli. Beni egbi kɛtsɔ ŋwɛi ŋtayaa nɔ lɛ, ajie nɔkwɛmɔnɔ lɛ awo 0.5% TFA kɛ 3% acetonitrile mli, ni atsɔ nanoflow RP chromatography ni akɛ mass spectrometry fata he lɛ nɔ akwɛ 500 ng mli kɛtsɔ gbɛjianɔtoo lɛ mli nibii ni atsɔɔ mli fitsofitso kɛtsɔ hiɛ lɛ nɔ.
Kɛ MaxQuant v.1.5.3.30 (56) atsu nii kɛha niyenii ni haa mɔ he jɔɔ lɛ kɛ eyibɔ ni akɛtao Rps. tsɛŋemɔ hela mli wala yibii ahe saji (www.uniprot.org/proteoms/UP000001426). Akɛ nibii ni haa mɔ he jɔɔ lɛ ewo ProteomeXchange Alliance lɛ mli kɛtsɔ PRIDE hefatalɔi nitoohe lɛ nɔ (http://proteomecentral.proteomexchange.org) yɛ nibii ni akɛyooɔ PXD020402 lɛ shishi.
Kɛha RPLC ni akɛ electrospray ionization mass spectrometry fata he lɛ mlipɛimɔ lɛ, asaa RC-LH1 nibii ni yɔɔ srɔtoi lɛ kɛjɛ koo mli Rps. Kɛtsɔ gbɛ ni akala tsutsu lɛ nɔ (16), protein ni ajieɔ lɛ kpo yɛ palustris mli lɛ ji 2 mg ml-1 yɛ 20 mM Hepes (pH 7.8), 100 mM NaCl kɛ 0.03% (w/v) β- (Bio-Rad mlipɛimɔ) ) DDM mli. Taakɛ mɔ ni fee lɛ mla lɛ tsɔɔ lɛ, okɛ 2D hetsemɔ nibii (GE Healthcare, USA) atsu nii ni okɛjie protein 10 μg kɛtsɔ nugbɔshiimɔ gbɛ nɔ, ni okɛ nugbɔshiimɔ lɛ awo 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile/ 20% vter (20%) mli. Akɛ RPLC (Dionex RSLC) ni akɛ tsɔne ni akɛkwɛɔ nibii amli lɛ (Maxis UHR-TOF, Bruker) fata he lɛ pɛi mli. 1.2×100 mm ŋwɛi ŋta (Thermo Fisher Scientific, UK) kɛha mligbalamɔ yɛ 60°C kɛ 100μlmin -1, kɛ ŋwɛi ŋta ni ji 85% (v / v) ŋta A [0.1% (v / v) FA kɛ 0.02% (V/v) ŋta B5 a 0.1%(v/v) FA kɛ 0.02%(v/v) yɛ 90%(v/v) acetonitrile TFA mli] Kɛ akɛ electrospray ionization jɛɛhe ni yɔɔ ŋmɛnɛŋmɛnɛ kɛ shishijee nibii ni akɛtsuɔ nii lɛ tsu nii fe minitii 60 lɛ, tsɔne ni akɛkwɛɔ nibii ni yɔɔ ŋwɛi lɛ amli lɛ naa 100 kɛyashi 2750 m/z (mass-to-charge ratio). Kɛtsɔ ExPASy wala he saji ahe nibii ni akɛtsuɔ nii yɛ FindPept dɛŋdade (https://web.expasy.org/findpept/) lɛ yelikɛbuamɔ nɔ lɛ, feemɔ shikpɔŋ he mfoniri ni kɔɔ ŋwɛibɔfoi lɛ ahe kɛya ŋwɛibɔfoi lɛ amli fãi bibii lɛ anɔ.
Akɛ ŋmɛlɛtswai 72 tsu nii yɛ 100 ml NF-shishijee (10μMm-2 s-1), teŋgbɛ (30μMm-2 s-1) loo nɔ ni nɔ kwɔ (300μMm-2 s-1) la shishi. M22 mli (M22 mli ni ajieɔ ammonium sulfate kɛjɛɔ mli ni akɛ sodium acetate yeɔ sodium succinate najiaŋ) yɛ 100 ml ni akɛ ŋmei woɔ nɔ (23) mli. Yɛ 30-s sɛɛkuu enumɔ mli lɛ, akɛ 0.1 micron glase ŋmeii wo mli yɛ 1:1 mli koni akɛfolɔ wala yibii lɛ amli ni akɛwo ais nɔ minitii enumɔ. Ajie nibii ni nyɛɛɛ atsɔ mli, wala yibii ni ekumɔ kɛ glase ŋmeii kɛtsɔ 16,000 RCF nɔ minitii 10 yɛ benchtop microcentrifuge mli. 100,000 RCF ni yɔɔ 20 mM tris-HCl (pH 8.0) ni 40/15% (w/w) sucrose ŋmɔlɔ yɔɔ mli lɛ mli ŋmɛlɛtswai 10.
Taakɛ atsɔɔ mli yɛ wɔ nitsumɔ ni eho lɛ mli lɛ, immunodetection ni ji E-tag ni yɔɔ PufW (16) lɛ nɔ lɛ. Yɛ kukufoo mli lɛ, ŋwɛi ŋta ni atsuu he (11.8 nM) loo ŋwɛi ŋta ni RC lɛ falɛ kome lɛ nɔŋŋ yɔɔ mli lɛ (ni atsɔɔ mli kɛtsɔ oksideshin ni jieɔ srɔtofeemɔ ni eba shi lɛ nɔ ni ekɛ jatsu ni yɔɔ gel ni awo mli muji lɛ nɔ lɛ kpãa gbee lɛ nɔ) yɛ 2x SDS loading buffer (Merck, UK) ni ajie mli shii enyɔ lɛ mli. Ajara niyenii mli wala yibii lɛ amli yɛ 12% bis-tris NuPage gel (Tɛrmo Fisher Jeŋ Nilee, UK) nɔ. Akɛ Coomassie Brilliant Blue (Bio-Rad, UK) wo gel ko mli koni akɛwo mli ni akɛna RC-L fã bibioo lɛ. Akɛ niyenii ni yɔɔ gel ni ji enyɔ lɛ nɔ lɛ tee tsofa ni haa mɔ he jɔɔ lɛ mli (PVDF) mli koni akɛkwɛ bɔ ni gbɔmɔtso lɛ mli nibii ni gbeɔ helai lɛ tsuɔ nii amɛhaa lɛ mli. mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 kɛ 5% (w / v) mliki ni ajie mli ŋmɔtɔ, ni no sɛɛ lɛ akɛ tsofa ni teɔ shi ewoɔ E-His shishijee tsofa lɛ wo mli (yɛ buffer tris-5l 1000 A190-114A, Bethyl Laboratories, U.S.A.) lɛ mli ŋmɛlɛtswai 4 sɔŋŋ. Beni afɔ he shii etɛ kɛ minitii enumɔ yɛ tsofa ni gbeɔ hela mli lɛ, akɛ tsofa ni gbeɔ hela ni ji horseradish peroxidase (Sigma-Aldrich, UK) lɛ fata he (ni akɛ 1:10,000 wo tsofa ni gbeɔ hela mli lɛ mli) Wo mli koni anyɛ ana (minitii enumɔ yɛ tsofa ni gbeɔ hela mli shii etɛ sɛɛ. (Cyanagen, Italy) kɛ Amersham Mfoniri 600 (GE Hewalɛ Hekwɛmɔ, UK).
Kɛtsɔ gel ni awo mli muji loo immunoassay gbɛ lɛ eko fɛɛ eko mli hewalɛ mlijaa lɛ nɔ lɛ, ni akɛ he ni yɔɔ ŋwɛi ŋta lɛ shishi lɛ afee ekome ni abu RC-L (gel ni awo mli muji) kɛ Protein-W (immunoassay) hewalɛ mlijaa lɛ he akɔntaa, yɛ ImageJ (57) Tsu mfoniri lɛ he nii. Atsake nɛkɛ mlijaa nɛɛ awo molar mlijaa mli kɛtsɔ susumɔ ni asusu akɛ mlijaa ni yɔɔ RC-L kɛ protein-W ni yɔɔ RC-LH114-W nɔkwɛmɔnɔ krɔŋŋ lɛ mli lɛ ji 1:1 ni akɛ saji fɛɛ ni ato lɛ fee bɔ ni sa lɛ nɔ.
Kɛji ootao nibii krokomɛi ni fata he kɛha niŋmaa nɛɛ, ofainɛ kwɛmɔ http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1.
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