Ubu†Aderesi y’ubu: OX11 0DE, Ubwongereza, Inyubakwa y’Idayimoni, Ibarabara ry’Ivya Siyansi n’Ibishasha rya Harwell, Dietcote, Oxfordshire, Ubwongereza, Ishirahamwe ry’Isoko ry’Umuco w’Idayimoni, Ltd., Ikigo c’Ibishushanyo vy’Ibinyabuzima vy’Ikoranabuhanga.
Igihimba c’umuco gikora 1 (RC-LH1) ni co gice nyamukuru c’imigera y’ibara ry’umuyugubwe. Twarashizeho imiterere ibiri y’imikorosikopi y’ubuhinga bwa none y’umugwi wa RC-LH1 uva ku gikoko citwa Rhodopseudomonas palustris. Igishushanyo c’uburebure bwa 2,65-Å c’umugwi wa RC-LH114-W gifise ibice 14 vy’umubiri bikikuje RC, ivyo bikaba bicikamwo na poroteyine W, mu gihe umugwi utagira poroteyine-W ari RC yose ikikujwe na RC. Ivyiyumviro 16 vy’uruzitiro LH1. Igereranyo ry’ivyo bihimba ritanga ubumenyi ku bijanye n’inguvu za quinone mu gisata ca RC-LH1, harimwo n’amahinduka y’imiterere atamenyekanye mbere igihe quinone ifatanya ku kibanza ca RC QB, hamwe n’aho ibibanza vy’ugufatanya quinone vy’inyongera biri, bifasha kubijana muri RC. Ubuhinga budasanzwe bwa poroteyine W burabuza ko uruzitiro rwa LH1 rufunga, gutyo bikaba bituma haba umurongo wo kwihutisha uguhinduranya quinone/quinolone.
Inguvu zitangwa n'izuba zishobora gutunga hafi ubuzima bwose bwo kw'isi, kandi zifise ubushobozi bwinshi mu vy'ubuhinga bw'ibinyabuzima bikoresheje izuba. Naho nyene zitera imbere mu bijanye n’uguhingura ibiribwa kw’isi yose, imigera y’ibara ry’umuyugubwe na yo nyene yerekana uburyo butandukanye bw’inguvu be n’ubushobozi bwo guhindura ibiribwa. Zishobora kwirinda gukora ibifungurwa biva ku zuba maze zigakura nk’imigera ifungura ibintu bitandukanye mu mwiza, zishobora gukosora azote n’umuyagankuba, zigatuma habaho ihydrogène, kandi zigasenyura ibintu bimota neza (1-3). Kugira ngo umuco ubone inguvu z’ivyo bikorwa, utegerezwa guhindurwa ningoga kandi neza ukagira inguvu z’imiti. Ivyo bitangura iyo igice c’antene gifata umuco gifata umuco kigaca kirungika inguvu zifashwe mu kibanza c’ugusubiza (RC), gutyo kigatangura gutandukanya ivy’umuyagankuba (4 – 7). Igihimba c’ishimikiro c’uguhingura umuco mu migera y’ibara ry’umuyugubwe igizwe n’ubwoko bwa 2 RC, ikikujwe n’umugwi 1 (LH1) wimbura umuco, kikaba ari co gikora umugwi nyamukuru wa RC-LH1. LH1 igizwe n’urutonde rw’ibihimba vy’umubiri bifise αβ bigoramye, kimwe cose muri vyo kikaba gifatanya n’ibihimba bibiri vy’umubiri bita chlorophylle (BChl) n’ibihimba bibiri vy’umubiri bita carotenoïdes (8-12). Igikoresho coroshe kuruta ibindi vyose gifise αβ heterodimers 16 canke 17 zikikuje RC (9-13) mu nzitiro yugarijwe, ariko mu bindi bihimba vy’umubiri, amapeptide agendagenda mu ruhu arahagarika ububandanya bwa LH1 ikikuje , Gutyo bigatuma habaho ugukwiragira kwa quinol/quinone na cytochle hagati ya bRC1c1. 13-15). Igiterwa c’ibara ry’umuyugubwe citwa Rhodopseudomonas (Rps.) ni ikinyabuzima c’akarorero gishobora gutahura inguvu n’uguhinduranya ama elegitoroni bifasha mu gukora ibikorwa vy’izuba. Igishushanyo ca mbere c’ikibiriti ca Rps. Igishushanyo c’umugwi wa palustris RC-LH1 ni RC, ukikujwe n’ibiziga 15 vy’ibihimba vy’umubiri bita LH1, ivyo bikaba bicikamwo n’igihimba kitazwi citwa “Protéine W” (14). Proteine-W yaje kumenyekana ko ari RPA4402, ari yo proteine ​​idafise ibimenyetso vy’uburemere bwa 10.5kDa ifise ibihimba bitatu vy’umubiri (TMH) (16). Turasaba guhindura izina ry’umugera wa rpa4402 ukoresha poroteyine W ngo ube pufW kugira ngo uhure n’amazina akoreshwa ku mugera ushiramwo poroteyine RC-L, M (pufL, pufM) na LH1α, β (pufA, pufB). Igitangaje ni uko proteine-W iri gusa mu bice nka 10% vya RC-LH1, igahishura Rps. palustris itanga ibihimba bibiri bitandukanye vy’umubiri bita RC-LH1. Aha, turatanga raporo y’imiterere y’ibihimba bibiri vy’umubiri (cryo-EM) vy’ibihimba bibiri vy’umubiri, kimwe gifise poroteyine W n’ibihimba 14 αβ bitandukanye, ikindi kitagira poroteyine W be n’uruzitiro rwugarijwe rwa 16 Heterodimer LH1. Igishushanyo cacu kigereranya ihinduka ry’intambwe mu gutahura RC-LH1 complex ya Rps. palustris, kuko twasuzumye igitigiri c’abantu bamwebamwe b’ubwoko bumwebumwe kandi dufise ubushobozi buhagije bwo gutanga neza peptide imwe imwe n’ibara ry’umubiri riboshe be n’amavuta n’ibindi bihimba bifitaniye isano n’ivyo. Igereranyo ry’ivyo bihimba vyerekana ko izo poroteyine zitatu za TMH-W zitabonetse mu yindi migwi yose ya RC-LH1 gushika ubu zitanga umurongo wa quinone kugira ngo zihutishe uguhinduranya quinone/quinolone. Igitigiri c’ibibanza vy’ugufatanya amavuta n’ibindi bihimba vy’umubiri vyazigamye vyaramenyekanye, kandi twahishuye ihinduka rishasha ry’imiterere inyuma y’uguhuza quinone na RC, rishobora kuba ryiza kuri photosystem II (PSII) RC y’ibinyabuzima bifungura umuco bifise umwuka w’ikirere. Ivyo twabonye biratanga ubumenyi bushasha ku bijanye n’ingene quinone/quinolone zifatanya n’ibindi bihimba vy’umubiri bifatanya n’ibindi bihimba vy’umubiri bita quinone mu gice ca RC-LH1 core complex c’imigera y’ibara ry’umuyugubwe.
Kugira ngo bishobore kworohereza inyigisho ido n’ido y’ivyo bibanza bibiri biboneka mu Rps. palustris, dutandukanya RC-LH1 imwe yose biciye ku buryo bw’ubuhinga bw’ibinyabuzima. Ico gice c’umubiri kidafise poroteyine W (ikizokwitwa ΔpufW) carasukuwe kiva ku bwoko butagira umugera wa pufW (16), kandi igihimba kimwe gusa c’umubiri citwa RC-LH1 ni co gishobora gukorwa. Ivyo bihimba birimwo poroteyine W biva ku bwoko bumwe. Proteine ​​W y’uwo mugera irahindurwa ikoresheje ikimenyetso ca 10x His ku mpera yayo C, kugira ngo iyo proteine ​​W irimwo ishobore gufatanywa neza na proteine ​​W nyinshi zidafise biciye mu gutuma ivyuma bidakora. Ivyo bihimba bitandukanye neza (16) Ivyuma bipima ibara ry’umubiri (IMAC).
Nk’uko vyerekanwa ku gishushanyo ca 1, izo nzira zompi zirimwo ibice bitatu vy’ubuhinga bwa none (RC-L, RC-M na RC-H) bikikujwe n’umurongo wa LH1. Igishushanyo ca 2.80-A c’ico gice kitagira poroteyine-W kirerekana αβ heterodimers 16, zikora uruzitiro rwa LH1 rwugarijwe rukikuje RC rwose, aha rero rukaba rwitwa igice ca RC-LH116. Igishushanyo ca 2,65 Å c’ivyo bihimba birimwo poroteyine-W gifise LH1 y’ibihimba 14 bitandukanye bicitsemwo na poroteyine-W, ubu bikaba vyitwa RC-LH114-W.
(A na B) Igishushanyo c’imbere c’ivyo bihimba. (C na D) Amabara afatanye yerekanwa mu nkoni. (E na F) Ivyo bihimba vy’umubiri vyerekanwa bivuye ku ruhande rw’umubiri bifise amapeptide n’ibihimba vy’umubiri bita LH1 bigereranywa mu bishushanyo vy’abantu, kandi biharurwa nk’uko isaha igenda biva ku nzira y’isaha kuva ku nzira y’amaproteine-W [bihuye n’uguharurwa kwa Rba. n’ibindi bihimba vy’umubiri (13)]. Ku bijanye na LH1-α, ibara ry’igice c’umubiri gifise poroteyine ni umuhondo; ku bijanye na LH1-β, ibara ry’igice c’umubiri gifise poroteyine ni ubururu; ku bijanye na poroteyine-W, iyo poroteyine iratukura; ku RC-H, ni cyan; ku RC-L, ni Orange; ku RC-M, ni umutuku w’agahama. Cofactors zigereranywa n’inkoni, icatsi kibisi kigereranya ama molecule ya BChl na BPh a, ibara ry’umuyugubwe rigereranya carotenoïdes, umuhondo na wo ugereranya ama molecule ya UQ10. (G na H) Igishushanyo kinini c’ikinogo ca poroteyine-W mu karere kangana k’umugwi wa RC-LH114-W (G) n’umugwi wa RC-LH116 (H). Cofactors zigaragazwa mu buryo bwo kwuzuza ikibanza, chelated quinone yerekanwa mu bururu. Ico kinogo ca proteine-W kigaragazwa n’umurongo w’ubururu ufise uturongo muri (G), kandi utunogo dutoduto aho quinone/quinolol ikwiragizwa ku mpeta ya LH116 tugaragazwa n’umurongo w’ubururu ufise uturongo muri (H).
Igishushanyo 1 (A na B) kirerekana RC ikikujwe n’imirongo yuguruye canke yugarijwe y’ibihimba vy’umubiri bita LH1αβ heterodimers, kimwe cose kikaba gifatanya BChl zibiri n’icarotenoid imwe (Igishushanyo 1, C na D). Ivyigwa vya kera vyerekanye ko Rps ari co gice ca LH1. Mu nzira y’ibinyabuzima vy’ibinyabuzima bita spirulina xanthin, ubwo bwoko burimwo ibinyabuzima bivanze vy’ibinyabuzima bita carotenoïdes (17). Ariko rero, spiropyrroxanthin ni yo ihambaye cane kandi uburemere bwayo buraryoshe. Ku bw’ivyo, twahisemwo gushushanya spiroxanthin ku bibanza vyose LH1 ifatanya. Ivyo bihimba vy’umubiri bita alpha na beta ni TMH zimwe zimwe zifise uturere two hanze tw’uruzitiro rugufi (Ishusho 1, A, B, E, na F). Naho uburemere bw’ibisigazwa 17 ku mpera ya C butabonetse, alpha polypeptide yaracitsemwo ibice kuva kuri Met1 gushika kuri Ala46 muri ivyo bihimba vyose bibiri. β polypeptide yagabanutse kuva kuri Gly4 gushika kuri Tyr52 muri RC-LH116, kuva kuri Ser5 gushika kuri Tyr52 muri RC-LH114-W. Nta n’uburemere bw’ibisigazwa 3 canke 4 vy’iherezo rya N canke 13 vy’iherezo rya C vyabonetse (Ishusho S1). Isesengurwa ry’uburemere bw’ibintu bivanze RC-LH1 ryateguwe bivuye ku bwoko bw’ibikoko ryerekanye ko ako karere kabuze kari ingaruka y’ugucapura kw’izo peptides (Ishusho S1 na S2). Ivyo bimenyetso vy’iherezo rya N vya α-Met1 na vyo nyene vyabonetse (f). Iryo sesangura ryerekanye ko α-peptide igizwe n’ibisigazwa fMet1 gushika kuri Asp42/Ala46/Ala47/Ala50, β-peptide na yo igizwe n’ibisigazwa Ser2 gushika kuri Ala53, ivyo bikaba bihuye neza n’ikarita y’uburemere bw’ubushuhe buke bwa EM.
Guhuza α-His29 na β-His36 bituma BChls zihura amaso mu yandi; buri heterodimere αβ ikoranira hamwe n’ababanyi bayo kugira ngo ikore uruzitiro rwuguruye (RC-LH114-W) canke uruzitiro rwugarijwe (RC-LH116) rukikuje RC Igishushanyo c’ibara ry’umubiri gifatanye n’ivyo bita exciton (Ishusho 1, C na D). Ugereranije n’umurongo wa 877 nm wa RC-LH114-W, umurongo w’umutuku w’ugufata 880 nm wa RC-LH116 ni 3 nm (Ishusho 2A). Ariko rero, uruzitiro rw’ibara ry’uruziga rwitwa dichroïsme spectrum ni hafi rumwe (Ishusho 2B), ivyo bikaba vyerekana ko naho hariho itandukaniro rigaragara hagati y’uruzitiro rwuguruye n’uruzitiro rwugarijwe, ibidukikije vyo mu karere ka BChls birasa cane. Ivyo bishobora kuba bivuye ku kugabanya ubushuhe n’ugushikama ku nzira yugarijwe (18, 19), ihinduka ry’ugufatanya kw’ibara ry’umubiri riterwa n’uruzitiro rwugarijwe (20, 21), canke ivyo bibiri bihurikiye hamwe (11).
(A) Ibara ry’umuco w’izuba/iboneka/iri hafi y’umuco w’izuba, isonga ryaryo rikaba ryashizweko ikimenyetso c’ibara ryaryo rihuye n’iryo kandi rikaba risanzwe ku isonga rya BPh ku 775 nm. (B) Ibara ry’uruziga ry’ibara ry’umubiri ry’uruziga ry’umubiri ry’umubiri ry’umubiri ry’umubiri ry’umubiri ry’umubiri. (C na D) Ivyiyumviro vya ΔA vyatowe mu bimenyetso vy’ugufata ibintu vy’igihe vy’umugwi wa RC-LH114-W (C) n’umugwi wa RC-LH116 (D). Kugira ngo bigereranywe neza, imirongo yose irasanzwe ku ∆A ya −A ku 0.2 ps. (E) Igipimo c’uguhinduka kw’imitsi ya cytochrome c2 inyuma y’uguterwa n’imishwarara iyo hariho ivyiyumviro bitandukanye vya UQ2 (raba igishushanyo S8 ku makuru atagiramwo ivyatsi). (F) Mu ngingo zakuriye mu muco w’uburemere buke, buringaniye canke bwinshi (10, 30 canke 300μMm-2 s-1, uko bikurikirana), poroteyine W na RC-L ziri mu gice c’umubiri gisukuye be n’igipimo c’uruzitiro rwatandukanye. Kumenya urugero rwa poroteyine hakoreshejwe ubuhinga bwa SDS-polyacrylamide gel n’ubuhinga bwo gupima ubudahangarwa bw’umubiri (raba igishushanyo S9 ku makuru atagiramwo ivyatsi). Menya igipimo kijanye n’igice ca RC-LH114-W casukuwe. Igipimo c'uburemere bwa RC-L na proteine-W y'ivyo bihimba ni 1:1.
BChls ziri mu kibanza ca 1 mu nzira y’ubumuga αβ14 ya RC-LH114-W (Ishusho 1, A, C, na E) ziri hafi y’umuterankunga w’intango wa RC (P) ku 6,8Å kuruta BChls zingana ziri muri RC-LH116 (Ishusho 1, B, S3); ariko, ubuhinga bwo gufata ibintu mu gihe gito bw’ivyo bihimba bibiri vyerekana ko kuri RC-LH114-W na RC-LH116, igihe co guhindura inguvu z’ugukangura kuva kuri LH1 gushika kuri RC ari 40 ±4 na 44±3 ps (Ishusho 2). , C na D, Igishushanyo S4 n’Imbonerahamwe S2). Nta tandukaniro rikomeye kandi riri mu gutanga amakuru biciye ku buhinga bwa none muri RC (Ishusho S5 n’ivyanditswe vy’inyongera bijanye). Twikeka ko uguhuza cane kw’igihe co gutanga inguvu hagati ya LH1 na RC-P biterwa n’urugendo, imfuruka n’inguvu zishobora gukoreshwa zisa n’iza BChl nyinshi mu nzira zibiri za LH1. Bisa n’uko gutohoza uburyo bw’inguvu za LH1 kugira ngo umuntu ashike ku ntambwe ntoyi cane bitanyaruka kuruta gukura ingufu zitaziguye zivuye mu bibanza bitari vyiza zija muri RC. Igipfukisho ca LH1 gifunguye muri RC-LH114-W gishobora kandi gushikirwa n’ubushuhe butari bwinshi mu bihe vy’ubushuhe buke kugira ngo hakorwe isesengura ry’imiterere, kandi hariho αβ14 ndende y’uruzitiro ku bushuhe bwo mu cumba kuva ku ntambwe y’ibara rya βBChls ku kibanza ca RC 1.
Ivyo bihimba vy’umubiri bita RC-LH116 birimwo BChls 32 n’ibihimba vy’umubiri 16 vyitwa carotenoïdes, kandi uburyo bitunganijwe muri rusangi ni nk’ubwo biva ku bihimba vy’umubiri bita Thermochromatium (Tch.) pidpidum [9), ivyatsi bita Thiorhodovibrio ID9T9r. (12) n’ibimera vy’ibitoke (Blc.viridis) (10). Inyuma y’ugutunganya, hariho gusa ukunyuranya gutoyi mu bibanza vy’ibihimba vy’umubiri bita αβ heterodimers, cane cane 1-5, 15, na 16 (Ishusho S6). Kuba hariho proteine-W biragira ingaruka zikomeye ku miterere ya LH1. Ivyo bihimba bitatu vy’umubiri bita TMH bihuzwa n’ibiziga bigufi, bifise iherezo rya N ku ruhande rw’uruzitiro rw’ico gice c’umubiri, iherezo rya C rikaba ku ruhande rw’uruzitiro rw’umubiri (Ibishushanyo 1A na 3, A gushika kuri D). Proteine-W ahanini irakunda amazi (Ishusho 3B), kandi TMH2 na TMH3 zikorana na LH1αβ-14 kugira ngo zibe igice c’imbere c’umubiri (Ishusho 3, B na E gushika kuri G). Ivyo bihimba vy’umubiri bifise ahanini ibisigazwa vya Phe, Leu na Val mu karere k’umubiri. Ivyo bisigi vy’amazi vyirundanijwe n’ibihimba vy’umubiri bitagira amazi be n’ibihimba vy’umubiri bita αβ-14. Hari n’ibisigazwa vy’ibice vy’umubiri bifasha mu gukorana, harimwo n’ubucuti bw’iyidrojeni buri hagati ya W-Thr68 na β-Trp42 ku ruhande rw’ico kinogo gikomeye (Ishusho 3, F na G). Ku ruhande rw’umubiri, Gln34 iri hafi y’umugwi wa keto w’ibihimba vy’umubiri bita αβ-14 carotenoïdes. Vyongeye, umugera wa n-dodecyl β-d-maltoside (β-DDM) waratowe umuti, kandi umurizo wawo udashobora guterwa n’amazi waraguye gushika ku nzira iri hagati ya proteine-W na αβ-14, kandi umurizo w’amavuta ushobora kuba uri mu mubiri. Twarabonye kandi ko uturere tw’ugutorera umuti C-terminal twa proteine ​​W na RCH turi hafi cane, ariko ntituri mu rugero rwo guhingura imigenderanire yihariye (Ishusho 1, A na E). Ariko rero, hariho ugukorana mu bihimba vy’umubiri bitagira umuti vy’izo poroteyine zibiri, ivyo bikaba bishobora gutanga uburyo bwo gutora poroteyine-W mu gihe c’iteraniro ry’umugwi wa RC-LH114-W.
(A) Protein-W, irabanye n’aho ihuriye na LH1αβ14 mu buryo bw’ibishushanyo, ifise uruzitiro rwo ku ruhande rumeze nk’inkoni (urutukura), rwerekanwa mu gice c’ikigereranyo c’ubushobozi bw’umuyagankuba (uburebure bw’umweru bubonerana bufise urugero rw’uruzitiro rwa 0,13). (B) Proteine-W igereranywa n’ibara ry’amazi ridakunda amazi. Ivyicaro vy’ibara ry’agahama n’ivy’ibara ry’agahama vyerekanywe mu rangi y’umutuku, ivyicaro vy’amazi vyerekanywe mu rangi y’umweru, ivyicaro vy’amazi bikomeye vyerekanywe mu rangi y’umuhondo. (C na D) Proteine-W igereranywa mu bishushanyo, iyo ihagaze ni nk’iyo muri (A) (C), kandi izunguruka 180° (D). Dushingiye ku kibanza kiri mu rutonde, ibisigazwa bishobora gutandukanywa bifata urutonde rw’ibara ry’umunywamazi, aho iherezo rya N ari ubururu, iherezo rya C rikaba umutuku. (E) Proteine-W mu buryo bumwe nk’ubwo muri (A), kandi ibisigarira biri ku nzira ya proteine-W:LH1 bigereranywa n’inkoni zifise ibimenyetso bifatanye. (F) Protein-W izunguruka 90° ugereranije na (E) na LH1αβ14 mu gishushanyo c’ibishushanyo, kandi ugereranije n’ibisigazwa vy’imbere mu gishushanyo c’imirongo. Ivyo bisigarira vy’ivyo bihimba vy’umubiri biva kuri beta polypeptide birashirwako ikimenyetso. Ico gikoresho kigaragazwa nk’umurongo uhuye n’ibara ry’igishushanyo ca 1, β-DDM yasenyutse yerekanwa n’ibara ry’umweru, umwuka w’ikirere na wo werekanwa n’umutuku. (G) Ivyo bigaragara muri (F) birahindurwa 180°, hakaba hariho ibisigarira bigaragara vy’ivyo bihimba vy’umubiri vyitwa alpha polypeptide.
Proteine-W isubirira αβ heterodimere (iya 15 iri ku gishushanyo 1F), gutyo igatuma uruzitiro rudafunga no guhengamira heterodimere zitatu za mbere za αβ. Vyabonetse ko mfuruka y’uguhengamira kw’ibara rya mbere ry’ibara ry’agahama ry’ibara ry’agahama ry’ibara ry’agahama ry’ibara ry’agahama ry’ibara ry’agahama ryari 25° gushika kuri 29° (Ishusho 1, A na E), ryari rigizwe n’ibara ry’agahama ry’ibara ry’agahama ry’i αβ-1 muri RC A sharp contrast-LH116 (Ishusho 1, B). Heterodimere ya kabiri n’iya gatatu zica ku 12° gushika ku 22° no ku 5° gushika ku 10°, uko zikurikirana. Kubera intambamyi ya steric ya RC, uguhengamira kwa αβ-1 ntikurimwo umugwi wa kabiri wa αβ (uwuhuye n'uwa 16 αβ ku gishushanyo 1F), gutyo bikaba bituma habaho ikinogo gitomoye mu mpeta ya LH1 (igishushanyo 1, A na E) . Kubera ko ata bihimba bibiri vy’umubiri bita αβ bitandukanye, biherekejwe n’ugutakaza BChl zine n’ibihimba bibiri vy’umubiri bita carotenoïdes, nta n’imwe muri izo carotenoïdes ifatanya n’igice ca αβ-1 gigoramye, ivyo bikaba bituma haba impeta ya LH114-W irimwo carotenoïdes 13 Vegetarian na 28 BChls. Ivyiyumviro vy'ivyo bihimba bibiri biri mu turere twa αβ1 gushika kuri 7 biri hasi y'ivyo mu bindi bice vy'uruzitiro rwa LH1, ivyo bishobora kwerekana uburemere bw'ibice vya LH1 biri hafi y'aho RC QB iri (Ishusho 4) .
Amashusho ya RC-LH114-W (A na B) na RC-LH116 (C na D) yerekanwa avuye ku nzira imwe yo hejuru/inyuma (A na B) (A na C) no ku ruhande rw’imyobo rw’igishushanyo 1. (B na D). Imfunguruzo zifise amabara zirerekanwa iburyo.
Iyindi nzira imwe rudende y’umubiri ifise igipimo c’uburemere bw’ibintu 1:14 ni igihimba c’umubiri citwa Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Ariko rero, proteine ​​W na PufX nta n’umwe agaragara ko asa, kandi zigira ingaruka zikomeye ku mibumbe yazo ya LH1. PufX ni TMH imwe ifise igice c’umubiri c’iherezo rya N gikorana n’uruhande rw’umubiri rw’igice ca RC-H (13) ku kibanza gihuye na Rps. 116αβ-16. PufX irema umurongo w’uguhinduranya quinone/kinolone hagati ya RC-LH1 n’umugwi wa cytochrome bcl kandi iri muri Rba zose. n’umubiri w’umubiri (13). Naho nyene urufatiro rw’ibintu bimwe bimwe ruri muri Rba. Igihimba c’umubiri citwa sphaeroides RC-LH1-PufX kiri ku kibanza co gufatanya poroteyine W muri RC-LH114-W, kandi ikinogo giterwa na PufX na poroteyine-W kiri ku kibanza kingana (Ishusho S7A). Ico kinogo kiri muri RC-LH114-W na co nyene gihuye n’umurongo w’ivyuma vyitwa quinone (8) wa Pseudomonas rosea LH1, ukaba ugizwe n’ibihimba bitagira isano na poroteyine W canke PufX (Ishusho S7B). Ikindi, umurongo wa quinone uri muri Blc. Igipande c’icatsi kibisi c’emeraude LH1 gikozwe mu gukuraho igice kimwe ca γ (7) kiri mu kibanza nk’ico (Ishusho S7C). Naho bihuzwa n’amaproteine ​​atandukanye, ukuboneka kw’izo nzira za quinone/quinolol mu kibanza kimwe mu gice ca RC-LH1 bisa n’akarorero k’uguhinduka kw’ibintu bihurikiye hamwe, bikaba vyerekana ko ikinogo caremwe na poroteyine W gishobora gukora nk’umurongo wa quinone.
Ico kinogo kiri mu nzira ya LH114-W gituma habaho akarere k’uruzitiro rukomeza hagati y’ikibanza co mu mutima c’uruzitiro rwa RC-LH114-W n’uruzitiro rwinshi (Ishusho 1G), aho guhuza izo nzira zibiri biciye mu nzira y’amaproteine ​​nk’uko biri mu maproteine. Igitero ca RC-LH116 gisa n’igice ca Tch gipfutse. Igikoresho kimeze nk’urushinge (22) (Ishusho 1H). Kubera ko ugukwiragira kwa quinone biciye mu nzira y’umubiri vyihuta kuruta ugukwiragira biciye mu muhora mutoyi w’amaproteine, uruzitiro rwa LH114-W rwuguruye rurashobora kwemera ko RC ihinduka vyihuta kuruta uruzitiro rwa LH116 rwugarijwe, kandi ugukwiragira kwa quinone mu RC bishobora kuba bifise uburenganzira buke. Kugira ngo tugerageze nimba poroteyine W igira ico ikoze ku guhindura quinones biciye muri RC, twakoze igerageza ry’uguhinduka kw’imitsi y’umubiri ku rugero runaka rwa ubiquinone 2 (UQ2) (ikigereranyo ca UQ10 kamere gifise umurizo mugufi w’isoprene) (Ishusho 2E). Naho kuba hariho quinone y’umubiri bishobora gutuma umuntu adashobora kumenya neza igitigiri c’ibintu bigaragara (RC-LH114-W na RC-LH116 bibereye 0,2±0,1μM na 0,5±0,2μM, hakurikijwe), igitigiri kinini cane c’ibintu vy’umubiri (RC-LH1±2.RC114-1). 28±5% ni nini kuruta RC-LH116 (3,6±0,1 e-RC-1 s-1).
Twabanje kugereranya ko proteine-W iri mu bice nka 10 kw’ijana vy’umubiri (16); aha, ibiharuro vy’ubuzima bw’ingingo z’ukukura zifise umuco muto, umuco uri hagati, n’umuco mwinshi ni 15±0,6%, 11±1% na 0,9±0,5, hakurikijwe % (Ishusho 2F). Igereranyo ry’ingero ry’ubuhinga bwa mass spectrometry ryerekanye ko kwongerako ikimenyetso ca histidine bitagabanyije ubwinshi bw’ivya proteine-W ugereranyije n’imigera y’ubwoko bw’ishamba (P = 0.59), rero ivyo bipimo si ikintu c’ubuhinga bwa proteine-W yahinduwe (Ishusho S10). Ariko rero, iyo nzira nkeyi y’ivyo bita proteine-W mu gisata ca RC-LH1 irashobora gutuma RC zimwezimwe zihinduka ku rugero rwihuta, gutyo bikagabanya uguhinduranya buhoro kwa quinone/quinolone mu gisata ca RC-LH116. Twabonye ko igitigiri kinini c’abafise umuco kidahuye n’amakuru aherutse gusohoka, ivyo bikaba vyerekana ko pufW gene expression yiyongera iyo hari umuco ukomeye (Ishusho S11) (23). Itandukaniro hagati y’uguhindura pufW n’ugushiramwo proteine-W mu gisata ca RC-LH1 riratera urujijo kandi rishobora kwerekana ukuntu iyo proteine ​​itunganijwe.
Mu RC-LH114-W, hariho imitsi 6 y’umutima (CDL), imitsi 7 y’umutima (POPC), imitsi 1 y’umutima (POPG) n’imitsi 29 y’umubiri (β-DDM) igenewe kandi igashushanirizwamwo imitsi 6 y’umutima, 24 y’umubiri, 2 y’imitsi y’umubiri n’imitsi 12. RC-LH116 (Ishusho ya 5, A na B). Muri izo nzira zibiri, CDL iri hafi ku ruhande rwa cytoplasme rw’ico gice, mu gihe POPC, POPG na β-DDM ziri ahanini ku ruhande rw’umuco. Ivyuma bibiri vy’amaraso n’ivy’ugusukura vyatanzwe mu karere ka αβ-1 gushika kuri αβ-6 k’umugwi wa RC-LH114-W (Ishusho 5A), bitanu na vyo vyatanzwe mu karere kangana na RC-LH116 (Ishusho 5B ). Ibindi bihimba vy’umubiri vyabonetse ku rundi ruhande rw’ico gice, ahanini CDL, vyirundanijwe hagati ya RC na αβ-7 gushika kuri αβ-13 (Ishusho 5, A na B). Ibindi bihimba vy’umubiri n’ivyo gusukura bishobora gutorwa mu buryo bw’imiterere biri hanze y’uruzitiro rwa LH1, kandi iminyororo y’amavuta y’umubiri ishobora gutorwa neza irashika hagati y’ibice vya LH1, bigaragazwa ko ari β-DDM muri RC-LH114-W, kandi bikaba bisobanurwa ko ari β-DDM muri RC A mixture of βPOPC-DHD16. Ivyicaro bisa n’ivyo vy’amavuta y’umubiri n’ivyo gusukura mu mibumbe yacu vyerekana ko ari ibibanza bifatanya bifitaniye isano n’umubiri (Ishusho S12A). Ivyicaro vy’ibihimba bingana muri Tch na vyo nyene birahuye neza. Umugwaneza na Trv. 970 RC-LH1s (Ishusho S12, B gushika E) (9, 12) n’ibisigazwa vy’ubufatanye bw’iyidrojeni vy’umugwi w’umutwe w’amaraso vyerekanye ko vyazigamye neza mu gukurikirana (Ishusho S13), bikaba vyerekana ko CDL yazigamye ifatanya na RC (24), ivyo bibanza bishobora kuba vyazigamye.
(A na B) RC-LH114-W (A) na RC-LH116 (B) peptides zigereranywa n’ibishushanyo, kandi amabara agereranywa n’inkoni, hakoreshejwe urutonde rw’amabara ruri ku gishushanyo ca 1. Amavuta yerekanwa n’umutuku, n’ibikoresho vyo gusukura vyerekanywe n’umweru. UQ iboshe ku bibanza vya RC QA na QB ni umuhondo, mu gihe UQ iri ukwayo ni ubururu. (C na D) Ivyiyumviro nk’ivyo (A) na (B), harimwo amavuta. (E gushika kuri G) Igishushanyo cagutse ca Q1(E), Q2(F) na Q3(G) bivuye kuri RC-LH116, bifise iminyororo yo mu ruhande ikorana. Ivyo bihimba vy’umubiri vyerekanywe nk’imirongo y’ibiharuro vyirabura.
Mu RC-LH116, RC QA na QB UQ zompi, zigira uruhara mu guhindura ama elegitoroni mu nzira yo gutandukanya ivyuma, zirabora aho zifatanya. Ariko muri RC-LH114-W, QB quinone ntiyatowe umuti kandi tuzobivugako mu buryo burambuye aha hepfo. Uretse QA na QB quinones, molecules zibiri za UQ zifise chelated (ziri hagati y’ibizingiti vya RC na LH1) zishirwa mu miterere ya RC-LH114-W hakurikijwe imigwi yazo y’imitwe yatowe umuti neza (ziri muri Q1 na Q2, uko zikurikirana). ikirere). Igishushanyo 5C). Ivyiyumviro bibiri vy’isoprene bishirwa kuri Q1, kandi ikarita y’uburemere itorera umuti imirizo 10 yuzuye ya isoprene ya Q2. Mu miterere ya RC-LH116, haciye hatorwa umuti ama molecule atatu ya UQ10 afise chelated (Q1 gushika kuri Q3, igishushanyo 5D), kandi ama molecule yose arafise uburemere bugaragara mu murizo wose (Ishusho 5, D gushika kuri G). Mu mibumbe ibiri, ivyicaro vy’imigwi y’imitwe ya quinone ya Q1 na Q2 birafise uguhuza neza cane (Ishusho S12F), kandi bikorana na RC gusa. Q1 iri ku bwinjiriro bw’ikinogo ca W ca RC-LH114-W (Ishusho 1G na 5, C, D na E), Q2 na yo iri hafi y’aho QB ifatanya (Ishusho 5, C, D) na F). Ivyo bisigazwa vya L-Trp143 na L-Trp269 vyazigamye biri hafi cane ya Q1 na Q2 kandi bitanga ubushobozi bwo gukorana π-stacking (Ishusho 5, E na F, n’Ishusho S12). L-Gln88, 3,0 Å kuva ku mwuka wo kure wa Q1, itanga ubucuti bukomeye bw’iyidorojeni (Ishusho 5E); iyo nsigarira irazigama mu RC zose kiretse ubucuti buri kure cane (Ishusho S13). L-Ser91 isubirizwa mu buryo bubereye Thr mu zindi RC nyinshi (Ishusho S13), ni 3.8 Angstroms iva ku mwuka wa methyl oxygen wa Q1, kandi ishobora gutanga amabondi y’ihydrogène agoyagoya (Ishusho 5E). Q3 ntiboneka ko ifise ugukorana kwihariye, ariko iri mu karere k’amazi hagati y’igice ca RC-M n’igice ca LH1-α 5 gushika ku 6 (Ishusho 5, D na G). Q1, Q2 na Q3 canke quinones chelates ziri hafi na zo nyene zaratowe umuti muri Tch. Umugwaneza, Trv. Ubwoko 970 na Blc. Igishushanyo c’iris (9, 10, 12) kirerekana ahantu h’infashanyo y’ugufatanya kwa quinone mu gisata ca RC-LH1 (Ishusho S12G). Ivyo bihimba bitanu vyaboze biri muri RC-LH116 birahuye neza n’ibice 5,8±0,7 vy’igice kimwekimwe cose kigenwa n’ubuhinga bwo gupima amazi (HPLC), mu gihe ivyo bihimba bitatu vyaboze biri muri RC-LH114-W biri hasi y’agaciro kagereranywa k’ibice 6,2±0,3 vyerekana ko hariho S4. ishusho.
Ivyo bihimba vy’umubiri bifise uburinganire bw’ibinyoma L na M, kimwe cose kirimwo TMH zitanu kandi gikora heterodimere ihuriza hamwe dimere imwe ya BChl, monomere zibiri za BChl, monomere zibiri za bacteriophage (BPh), n’icuma kimwe kitari Heme n’umugera umwe canke ibiri wa UQ10. Biciye mu kubaho kw’imigenderanire y’iyidrojeni ku mugwi wa ketone w’iherezo n’ukwirundanira kwayo kuzwi muri Rps, carotenoïdes zija mu gice ca M, citwa cis-3,4-dehydroorhodopin. Ubwoko (25). Igihugu c’inyuma c’uruzitiro rwa RC-H gifatanywa n’uruzitiro rwa TMH rumwe. Ubuhinga bwose bwa RC busa n’ubw’ibice bitatu vya RC vy’ubwoko bufitaniye isano (nka Rba). n’ibindi bikoko (Indangamuntu ya PDB: 3I4D). Ivyuma bikomeye vya BChl na BPh, umugongo wa carotenoid n’icuma kitari heme birahurirana mu rwego rw’ugutorera umuti kw’ivyo bihimba, nk’uko nyene bimeze ku mugwi w’umutwe wa UQ10 ku kibanza ca QA na QB quinone ku RC-LH116 (Ishusho S15).
Kuba hariho imibumbe ibiri ya RC ifise ibiharuro bitandukanye vy’abantu bafata ahantu ha QB bitanga akaryo gashasha ko gusuzuma amahinduka adahinduka y’imiterere ajana n’ugufatanya kwa QB quinone. Mu gisata ca RC-LH116, quinone ya QB iri mu kibanza “co hafi” kiboshe neza (26), ariko ugutandukanya kwa RC-LH114-W nta quinone ya QB igira. Nta quinone ya QB iri muri RC-LH114-W, ivyo bikaba bitangaje kuko iyo nzira ikora, kuruta iyo nzira ya RC-LH116 ifise QB quinone itorewe umuti mu buryo bw’imiterere. Naho izo mpeta zibiri za LH1 zifise ama quinones atandatu, zitanu zitorera umuti mu mpeta ya RC-LH116 yugarijwe, mu gihe zitatu gusa zifise aho zigarukira mu mpeta ya RC-LH114-W yuguruye. Ivyo bishobora kwerekana ko ibibanza vya RC-LH114-W QB bishobora gusubirizwa ningoga, ko quinone zikora cane mu gice gikomeye, kandi ko bishobora kujabuka uruzitiro rwa LH1. Turasaba ko ukubura UQ mu kibanza ca RC QB ca RC-LH114-W bishobora kuba ari ingaruka y’ikintu gikomeye cane kandi gikora cane, kandi ikibanza ca QB ca RC-LH114-W caraciye gica gikonja mu guhindura UQ Intambwe yihariye (injira mu kibanza ca QB yafunzwe a the conformation ref.
Iyo ata QB, uguhinduka kujana na L-Phe217 gushika mu kibanza kidahuye n’ugufatanya kwa UQ10, kuko bizotuma haba uguhura kw’ahantu n’igice ca mbere c’isoprene c’umurizo (Ishusho 6A). Ikindi, amahinduka agaragara y’imiterere y’ibintu aragaragara, cane cane helix de (helix ngufi mu nzira iri hagati ya TMH D na E) aho L-Phe217 yimurirwa mu mufuko wo gufatanya QB n’uguhinduka kwa L-Tyr223 (Ishusho 6A ) Kugira ngo uce ubucuti bw’ihydrogène n’ubufatanye bwa QB n’ubufatanye bwa M-4. ahantu (Ishusho 6B). Helix de izunguruka ku musozo wayo, Cα ya L-Ser209 ihinduka 0,33Å, mu gihe L-Val221Cα ihindurwa 3,52Å. Nta mpinduka ziboneka muri TMH D na E, zishobora gushirwa hejuru mu mibumbe yompi (Ishusho 6A). Nk’uko tubizi, iyi ni yo nzira ya mbere muri RC kavukire ifunga ikibanza ca QB. Ugereranije n’imiterere yuzuye (QB-bound) vyerekana ko imbere y’uko quinone igabanywa, bisaba ko habaho ihinduka ry’imiterere kugira ngo yinjire muri quinone. L-Phe217 irazunguruka kugira ngo ikore π-stacking interaction n’umugwi w’umutwe wa quinone, maze helix igahindukira ikaja hanze, igatuma umubiri wa L-Gly222 n’uruzitiro rw’inyuma rwa L-Tyr223 bikora uruzitiro rw’imigenderanire y’iyidrojeni rufise imiterere y’imigenderanire y’iyidorojeni ihamye (Ishusho 6, A na C).
(A) Igishushanyo c’ibara ry’agahama (uruzitiro rwa L, uruzitiro rwa orange/M, magenta) n’ibara ry’agahama (ibara ry’agahama), aho ibisigarira vy’ingenzi vyerekanywe mu buryo bw’ikigereranyo kimeze nk’inkoni. UQ10 igereranywa n’umurongo w’umuhondo. Umurongo w’utudomo werekana imigenderanire y’iyidrojeni iba mu mibumbe yose. (B na C) Igishushanyo c’imbere c’iyi apolipoproteine ​​n’imiterere yose y’impeta, kigaragaza umwuka w’uruzitiro rw’inyuma rwa L-Phe217 mu bururu na L-Tyr223 mu mutuku, uko bikurikirana. Igipande ca L ni umutuku; ibice vya M na H ntibigira ibara. (D na E) Ivyicaro vya RC QB (D) n’ivy’ivyatsi vyose (E) [ibara ry’ivyatsi (A) hakurikijwe] n’ivy’ivyatsi bitoto, ubururu n’ivyuma vy’ivyatsi vy’ivyatsi; ID ya PDB: 3WU2) Gushiramwo (58).
Mu buryo butategerezwa, naho hariho imiterere myinshi y’ibihimba vy’umubiri bifise ubukene bwa QB ata LH1, amahinduka y’imiterere yabonywe muri iki cigwa ntiyari yaravuzwe mbere. Ivyo birimwo n’imiterere y’ugukuraho QB iva kuri Blc. viridis (Indangamuntu ya PDB: 3PRC) (27), Tch. igiti c’umubiri (ID ya PDB: 1EYS) (28) na Rba. sphaeroides (PDB ID: 1OGV) (29), vyose bikaba bimeze hafi nk’imiterere yavyo yose ya QB. Igihe umuntu asuzumye neza 3PRC, yasanze amamoleke y’ivyo gusukura LDAO (Lauryl Dimethyl Amine Oxide) afatanya ku bwinjiriro bw’aho QB iri, ivyo bikaba bishobora gutuma adasubira gutunganya ibintu mu buryo bupfutse. Naho LDAO itabora mu kibanza kimwe muri 1EYS canke 1OGV, izo RC zitegurwa hakoreshejwe igikoresho kimwe co gusukura rero zishobora gutuma habaho ingaruka nk’izo. Igishushanyo c’ikibiriti ca Rba. Sphaeroides RC ifatanye n’ibara ry’umubiri c2 (PDB ID: 1L9B) na yo nyene isa n’iyifise ikibanza ca QB gipfutse. Ariko rero, muri iki gihe, akarere ka N-terminal ka RC-M polypeptide (gakorana n’aho QB ifatanya biciye ku nzira ya H y’ibisigazwa vya Tyr ku nzira ya Q) gafata uburyo butari ubw’akameremere, kandi ihinduka ry’uburyo bwa QB ntiryisuzumwa (30 ). Igihumuriza ni uko tutabonye ubwo bwoko bw’uguhinduka kw’umubiri w’umubiri wa M mu miterere ya RC-LH114-W, ukaba ungana n’akarere ka N-terminal ka RC-LH116 RC. Birakenewe kandi kumenya ko inyuma yo gukuraho antene ya LH1 ishingiye ku bikoresho vyo gusukura, RCs za apolipoproteine ​​ziri muri PDB zari zatowe umuti, ivyo bikaba vyakuyeho ibidengeri vy’imbere vy’ivyo bita quinone n’amavuta yo mu mutima mu kinogo kiri hagati ya RC n’imbere mu gice c’imbere c’uruzitiro rwa LH1 rukikuje (31, 32). RC iguma ikora kuko igumya ibintu vyose bifasha mu gukora, kiretse QB quinone ishobora kubora, iyo na yo ikaba idashikama cane kandi akenshi irazimangana mu gihe c’ugutegura (33). Ikindi, birazwi ko gukuraho LH1 n’amavuta y’umubiri agendagenda mu RC bishobora kugira ingaruka ku bikorwa, nk’ugugabanya ubuzima bw’umubiri utandukanijwe n’umuriro P+QB-state (31, 34, 35). Ku bw’ivyo, turakeka ko ukubaho kw’uruzitiro rwa LH1 rwo mu karere rukikuje RC bishobora kubungabunga ahantu ha QB “hafunze”, gutyo bikabungabunga ibidukikije vyo mu karere hafi y’aho QB iri.
Naho apolipoproteine ​​(ata QB quinone) n’imiterere yose bigereranya gusa amafoto abiri y’uguhinduka kw’aho QB iri, aho gushushanya urutonde rw’ibintu, hari ibimenyetso vyerekana ko iyo nzira yo gufatanya ishobora gukingirwa kugira ngo ntihagire isubira gufatanya na hydroquinone Kugira ngo ihagarike inhibition y’ivyo bihimba. Ugukorana kwa quinolol na quinone hafi y’aho QB iri apolipoproteine ​​bishobora kuba bitandukanye, ivyo bikaba bituma RC yanka. Haciye igihe kirekire bivugwa ko amahinduka y’imiterere afise uruhara mu gufatanya no kugabanya quinones. Ubushobozi bw’ama RC yakonje bwo kugabanya quinones inyuma yo kwimenyereza umwiza buragabanuka (36); Ivyerekana ko ivyo vyangiritse biterwa n’uko QB quinones zifatwa mu nzira “y’inyuma” nk’ibice 4,5 Å uvuye ku kibanza c’inyuma gikora (26) , 37). Turasaba ko iyo nzira y’ugufatanya kure ari ishusho y’ivyo hagati ya apolipoproteine ​​n’imiterere y’uruzitiro rwuzuye, bikurikira ugukorana kwa mbere na quinone n’ugufungura ahantu ha QB.
Ubwoko bwa II RC buboneka mu gisata ca PSII c’imigera imwe imwe ifungura amatara n’imigera y’ibimera, ibimera vy’ibitoke n’ibimera birafise uburinzi bw’imiterere n’imikorere (38). Ugutunganya imiterere yerekanwa ku gishushanyo ca 6 (D na E) gushimika ku gusa hagati y’ibihimba vya PSII RC n’aho QB y’umugwi wa RC w’imigera. Ukwo kugereranya kwamaze igihe kirekire ari akarorero ko kwiga uburyo bufitaniye isano cane n’ugufatanya n’ugugabanya kwa quinone. Ivyanditswe vya kera vyerekanye ko amahinduka y’imiterere ajana n’ugugabanya PSII kw’ibinyabuzima bita quinones (39, 40). Ku bw’ivyo, turavye ukuntu RC izigama mu buryo bw’iterambere, ubwo buryo bwo gufatanya butabonetse mbere burashobora no gukoreshwa ku kibanza ca QB ca PSII RC mu bimera bifungura umuco bifise umwuka w’ikirere.
Rps ΔpufW (ugukuraho kwa pufW kutagira ikimenyetso) na PufW-His (iherezo rya C 10x ry’ikimenyetso ca proteine-W igaragazwa n’aho pufW igaragara). palustris CGA009 vyari vyaradondowe mu gikorwa cacu c’imbere (16). Ivyo bikoko n’umuvyeyi w’ubwoko bw’ishamba vyavuye mu gikoresho co gukonjesha mu gucapura utugingo ngengabuzima dutoduto kuri PYE (buri 5 g litiro -1) (bibitswe muri LB ku -80 °C, birimwo 50% ( w/v) glycerol) proteine, umusemburo n’umusemburo (% succinate) agar/15. Iryo sahani ryashizwe mu mwiza ijoro ryose mu bushuhe bwo mu cumba mu bihe bitagira umwuka, hanyuma rica rimurikwa n’umuco w’umweru (~50 μmolm-2 s-1) watanzwe n’amatara ya OSRAM 116-W halogen (RS Components, UK) imisi 3 gushika kuri 5 gushika aho igikoko kimwe kibonetse. Igikoko kimwe carakoreshejwe mu gucandaga militiro 10 z’umuti wa M22+ (41) wongereweko 0,1% (w/v) y’aside za casamino (ubu zizokwitwa M22). Ico giterwa cakuriwe mu bihe vy’umwuka mutoyi mu mwiza ku bushuhe bwa 34°C hakoreshejwe uguhungabana ku 180 rpm mu kiringo c’amasaha 48, hanyuma 70 ml y’ico giterwa icandwa mu bihe nk’ivyo nyene mu kiringo c’amasaha 24. Igikoko gifise uburemere bwa militiro 1 gikoreshwa mu gucandaga militiro 30 z’umuti wa M22 mu cupa ry’ibirahuri ry’imiliyoni 30 ry’ibara ry’agahama ry’ibara ry’agahama ry’ibara ry’agahama, rikagira imishwarara n’uguhungabana (~50μmolm-2 s-1) mu kiringo c’amasaha 48 n’inguvu z’umuyagankuba zidashobora gutera Stirring rod. Hanyuma 30 ml y’ivyo bihimba vy’umubiri vyaciwemwo nk’ilitiro 1 y’ivyo bihimba vy’umubiri mu bihe nk’ivyo, ivyo bikaba vyaciye bikoreshwa mu gucandaga litiro nka 9 z’ivyo bihimba vy’umubiri vyamuritswe ku ~200 μmolm-2 s-1 mu kiringo c’amasaha 72. Ivyo bihimba vy’umubiri vyarimbuwe biciye mu gucapura ku 7132 RCF mu minota 30, bisubira gushirwa mu ~10 ml ya 20 mM tris-HCl (pH 8.0), hanyuma bibikwa ku -20°C gushika bikenewe.
Inyuma yo gufukura, wongeremwo amakirisitayo y’ibinyabuzima bimwebimwe vy’ibinyabuzima bita deoxyribonuclease I (Merck, mu Bwongereza), lysozyme (Merck, mu Bwongereza) n’ibipande bibiri vy’ibinyabuzima bita deoxyribonuclease I (Merck, mu Bwongereza) ku nsoro zisubiye guhagarara. Mu gice c’umuvuduko w’amazi c’Ubufaransa gifise ubushobozi bwa 20.000 psi (Aminco, muri Leta Zunze Ubumwe za Amerika), ivyo bihimba vyarahungabanijwe incuro 8 gushika ku 12. Amaze gukuraho utugingo ngengabuzima tutavunitse n’ibisigazwa bitashobora gusenyuka biciye mu gucapura ku 18.500 RCF mu minota 15 ku 4°C, iyo membrane yaravuye mu lysate y’ibara biciye mu gucapura ku 113.000 RCF mu masaha 2 ku 43.000°C. Ita igice gishobora gusenyuka hanyuma wongere ushire uruzitiro rw’ibara mu militiro 100 gushika kuri 200 za tris-HCl 20 mM (pH 8.0) hanyuma ushiremwo ibintu bimwe gushika ata bihimba biboneka. Ico gice c’umubiri cari gihagaze cashizwe mu mazi y’ibice 20 mM (pH 8,0) (Anatrace, USA) arimwo 2% (w/v) ya β-DDM mu kiringo c’isaha 1 mu mwiza ku bushuhe bwa 4°C hakoreshejwe uguvyurwa buhoro buhoro. Hanyuma ushiremwo centrifuge ku 70°C kugira ngo RCF 150.000 zisenyuke ku 4°C mu kiringo c’isaha 1 kugira ngo ukuremwo ibisigarira bidashobora gusenyuka.
Ivyo bihimba vy’umubiri biva ku bwoko bwa ΔpufW vyashizwe ku nkingi y’uguhindura ion y’imiliyoni 50 ya DEAE Sepharose ifise imirongo itatu y’inkingi (CV) y’ivyo gufatanya [20 mM tris-HCl (pH 8.0) irimwo 0.03% (w / v) β-DDM]. Woze iyo nkingi n’ibikoresho bibiri bifatanya CV, hanyuma woze iyo nkingi n’ibikoresho bibiri bifatanya birimwo NaCl 50 mM. Igitera RC-LH116 carakuwemwo n’umurongo w’ibara ry’umubiri ry’imirongo 150 gushika kuri 300 mM NaCl (mu nzira y’ugufatanya) ku 1,75 CV, igihimba c’umubiri gisigaye carasukuwe n’igikoresho co gufatanya kirimwo 300 mM NaCl ku 0,5 CV. Ikoranirize hamwe umurongo w’ugufata hagati ya 250 na 1000 nm, ugumye igice gifise igipimo c’ugufata (A880/A280) kirenze 1 ku 880 na 280 nm, ugishiremwo incuro zibiri mu buffer y’ugufatanya, hanyuma wongere ukoreshe uburyo bumwe ku nkingi ya DEAE Ku gusukura. Gucapura imice ifise ibiharuro vya A880/A280 birenze 1,7 n’ibiharuro vya A880/A805 birenze 3,0, ukore urugendo rwa gatatu rw’uguhindura ion, hanyuma ugumye ibice bifise ibiharuro vya A880/A280 birenze 2,2 n’ibiharuro vya A880/A805 birenze.0. Ivyo bihimba vy’umubiri vyari vyarasukuwe bimwe bimwe vyari vyinjijwe ku ml ~2 mu gicuncuro c’uburemere bw’amamolekule 100.000 (MWCO) (Merck, UK), hanyuma bishirwa ku nkingi y’ugukuraho y’ubunini bwa Superdex 200 16/600 (GE Healthcare, US) irimwo ubucunguzi bwa NaM thelume mu 200 m thelun 1.5 Igitabo c'ubuzima. Ikoranirize hamwe imirongo y’ugufata y’igice c’ugukuraho ubunini, maze ushire umutima ku mirongo y’ugufata n’imigereranyo A880/A280 hejuru ya 2.4 na A880/A805 hejuru ya 5.8 gushika kuri 100 A880, hanyuma uyikoreshe ubwo nyene mu gutegura canke kubika uruzitiro rwa cryo-TEM bikenewe Gumana kuri -8.
Igipande c’ivyatsi biva mu bwoko bwa PufW-His cashizwe ku nkingi ya Sepharose y’imililitiro 20 (tris-HCl 20 mM (pH 8,0) irimwo NaCl 200 mM na 0,03% (uburemere/uburemere)) mu gikoresho co gukingira ubuzima ( GE). v) β-DDM]. Iyo nkingi yayozwe n’ibice bitanu vy’umuti witwa IMAC, hanyuma n’ibice bitanu vy’umuti witwa IMAC birimwo histidine 10 mM. Ivyo bihimba vy’umubiri vyaciwe mu nkingi hakoreshejwe ibikoresho bitanu vy’ubuzima vyitwa IMAC birimwo histidine 100 mM. Ico gice kirimwo RC-LH114-W gishirwa hamwe kugera kuri ~10 ml mu gitereko c’amazi gifise akayunguruzo ka Amicon 100.000 MWCO (Merck, UK), kigavanwa incuro 20 n’umuti wo gufatanya, hanyuma kigashirwa ku militiro 25 Mu gitereko ca DEAE b imbere y’igihe. Woze iyo nkingi n’ibikoresho bine bifatanya CV, hanyuma ushiremwo ivyuma bifatanya CV umunani ku nzira y’umurongo iva kuri 0 gushika kuri 100 mM NaCl (mu bifatanya CV), hanyuma ushire ivyuma bine bisigaye birimwo ivyuma bifatanya 100 mM Ivyo bihimba vyasigaye vya chloridium chlorium/000. 2.4 n’igipimo ca A880/A805 kiri hejuru ya 4.6 imice yari yibanze gushika ku ~2 ml mu gicuncuro c’amazi c’i Amicon 100.000 MWCO, kandi yuzuye 1.5 CV IMAC imbere y’igihe Igicuncuro ca Superdex 200 cashizwe mu 16/60 1.5 Igitabo c'ubuzima. Ikoranye imirongo y’ugufata y’ibice vy’ubunini-bikuwemwo maze ushire imirongo y’ugufata n’ibiharuro A880/A280 hejuru ya 2.1 na A880/A805 ibiharuro hejuru ya 4.6 gushika kuri 100 A880, ivyo bikaba bikoreshwa ubwo nyene mu gutegura uruzitiro rwa TEM rwakonje canke bikabikwa ku -80°C.
Igikoresho co kwibiza mu mazi citwa Leica EM GP carakoreshejwe mu gutegura amabarabara ya TEM y’ubushuhe buke. Ivyo bihimba vyaciwe mu mazi ya IMAC gushika kuri A880 ya 50, hanyuma 5μl bishirwa ku gicapo c’umuringa gishasha c’umuringa QUANTIFOIL 1.2/1.3 (Agar Scientific, UK). Ushire iyo nzira ku bushuhe bwa 20°C n’ubukonje bwa 60% mu kiringo c’amasegonda 30, hanyuma uyipfuke mu masegonda 3, hanyuma uyizimye mu mazi ya ethane ku bushuhe bwa -176°C.
Amakuru y’ikigo RC-LH114-W yanditswe kuri eBIC (Ikigo c’Ivyuma vy’Ibinyabuzima) (Isoko y’Umuco w’Idayimoni y’Ubwongereza) hakoreshejwe igikoresho citwa Titan Krios, gikora ku nkomezi zinyaruka 300kV, gifise ubushobozi bwo gukura kw’izina bwa Choose ga.000 of-2 Igikoresho ca Gatan 968 GIF Quantum gifise igikoresho co kumenya isonga rya K2 carakoreshejwe mu kwandika amashusho mu buryo bwo kubara kugira ngo hakusanyirizwe amakuru. Ingano y'ibipimo ni 1.048Å, kandi igipimo c'imiti ni 3.83 e-Å-2s-1. Yakoranije iyo filime mu masegonda 11 maze ayigabanyamwo ibice 40. Koresha aho hantu harimwo carbone kugira ngo wongere ushire ahabona iyo mikorosikope, hanyuma ushire hamwe amasinema atatu ku mwobo umwe umwe. Muri rusangi, amasinema 3130 yarakoranijwe, afise agaciro ka defocus kari hagati ya -1 na -3μm.
Amakuru yerekeye iyo RC-LH116 yaronkejwe hakoreshejwe iyo mikorosikope nyene mu laboratwari y’ibinyabuzima y’i Asterbury (Kaminuza ya Leeds, mu Bwongereza). Amakuru yashizwe hamwe mu buryo bwo kubara n’ugukura kwa 130 k, kandi ubunini bwa pixel bwagereranywa na 1.065 Å n’urugero rwa 4.6 e-Å-2s-1. Iyo filime yafashwe mu masegonda 12, igabanywamwo ibice 48. Muri rusangi, amasanamu 3359 yarakoranijwe, afise agaciro k’ugutahura hagati ya -1 na -3μm.
Ivyo gukora amakuru vyose bikorwa mu muyoboro wa Relion 3.0 (42). Koresha Motioncorr 2 (43) kugira ngo ukosore inyigisho y’umurongo ukoresheje uburemere bw’imiti, hanyuma ukoreshe CTFFIND 4.1 (44) kugira ngo umenye umurongo wa CTF (igikorwa co guhindura ubudasa). Amafoto agaragara inyuma y’izo ntambwe z’intango zo gutunganya yerekanwa ku gishushanyo ca 2. S16. Ico kigereranyo c’uguhitamwo gica kiva mu guhitamwo n’amaboko nk’ibipimo 250 vy’ibice 1000 mu gicapo c’ibipimo 250 kandi ata n’imwe igaragara mu gushikiriza ibice bibiri (2D), gutyo hakanka ivyo bice bihuye n’ukwanduzwa kw’akarorero canke bitagira ibiranga bigaragara. Hanyuma, haciye hakorwa uguhitamwo kwikora ku mafoto yose matomato, kandi RC-LH114-W yari ibice 849.359, RC-LH116 na yo ikaba yari ibice 476.547. Ivyo bihimba vyose vyatowe vyaciye mu bice bibiri vy’ugushikiriza 2D bitagira ishingiro, kandi inyuma y’urugendo rwose, ibihimba bihuye n’aho carbone iri, ubuhumane bw’urugero, nta bimenyetso bigaragara canke ibihimba bifatanye cane biranka, bikaba bituma hakoreshwa 772.033 (90.9%) na 359.6578 vy’ibice3 (D) RC-LH114-W na RC-LH116 ni vyo bikurikirana. Igishushanyo c’intango c’ubuhinga bwa 3D cakozwe hakoreshejwe uburyo bwo kumanuka kw’ibarabara ry’ibarabara ry’ibarabara. Hakoreshejwe ikigereranyo ca mbere nk’ikigereranyo, ivyo bihimba vyatowe bishirwa mu mice ine mu buryo bwa 3D. Ukoresheje ikigereranyo kiri muri iki kiciro nk’ikigereranyo, ukore ivy’ugusukura 3D ku bihimba biri mu kiciro kinini kuruta ibindi vyose, hanyuma ukoreshe akayunguruzo k’intango ka 15Å low-pass kugira ngo ufunge ahantu h’umunyuro, wongereko amapixels 6 y’imirongo yoroshe, hanyuma ukoreshe amapixels kugira ngo ukosore Gatan K2 transfer function the top of Mo. Ku bijanye n’amakuru ya RC-LH114-W, iki kigereranyo ca mbere carahinduwe mu gukuraho uburemere bukomeye ku nkengera z’igipfukisho (butandukanye n’uburemere bukomeye bw’umubiri mu UCSF Chimera). Ivyo bigereranyo biva muri ivyo (ibipimo vya RC-LH114-W na RC-LH116 ni 3,91 na 4,16 Å, uko bikurikirana) bikoreshwa nk’ikigereranyo c’urugendo rwa kabiri rw’ugushikiriza 3D. Ivyo bihimba bikoreshwa bishirwa mu mugwi w’intango wa 3D kandi ntibirimwo isano rikomeye n’ababanyi. Gusangira canke kubura ibiranga imibumbe bigaragara. Inyuma y’urugendo rwa kabiri rw’ugushikiriza 3D, hatoranywe urutonde rufise ubushobozi bwo gutahura cane [Kuri RC-LH114-W, urutonde rumwe ni ibice 377.703 (44,5%), ku RC-LH116, hariho ibice bibiri gusa, vyose hamwe ni ibice 260.752 (% the W4d). inyuma y’uguhinduranya kwa mbere n’itandukaniro ritoyi]. Ivyo bihimba vyatowe birasubirwamwo mu gasandugu k’amapixel 400 hanyuma bikatunganywa hakoreshejwe ubuhinga bwa 3D. Igipfukisho c’umunyu gikorerwa hakoreshejwe akayunguruzo k’intango k’uburebure bwa 15Å, ukwaguka kw’ikarata y’ibipimo 3 n’igipfukisho c’ibipimo 3 vyoroshe. Hakoreshejwe ugutunganya CTF ku gice kimwekimwe cose, ugukosora inyigisho y’igice kimwekimwe cose n’urugendo rwa kabiri rw’ugutunganya CTF ku gice kimwekimwe cose, ugutunganya 3D, gupfuka ivyuma biva mu mazi n’ugusubiramwo ivy’inyuma birakorwa inyuma y’intambwe yose kugira ngo bikomeze gutunganya igihimba kivuyemwo. Hakoreshejwe agaciro k’iherezo ka FSC (Ikigereranyo c’Isano ry’Igikoko ca Fourier) ka 0,143, ivyerekanwa vy’ibigereranyo vya nyuma vya RC-LH114-W na RC-LH116 ni 2,65 na 2,80Å, hakurikijwe. Igishushanyo ca FSC c'ikigereranyo ca nyuma kirerekanwa ku gishushanyo ca 2. S17.
Urutonde rw’amaproteine ​​rwose rukurwa kuri UniProtKB: LH1-β (PufB; ID ya UniProt: Q6N9L5); LH1-α (Igikoko; Igikoko kimwe: Q6N9L4); RC-L (Igihugu; Indangamuntu y’Igihugu: O83005); RC-M (Igihugu; Indangamuntu y’Igihugu: A0A4Z7); RC-H (PuhA; Indangamuntu y’Igihugu: A0A4Z9); Igitera-W (PufW; Indangamuntu y’Igitera: Q6N1K3). SWISS-MODEL (45) yakoreshejwe mu kwubaka ikigereranyo c’ubumwe bwa RC, kirimwo urutonde rwa poroteyine za RC-L, RC-M na RC-H be n’imiterere y’ikibiriti ca Rba. sphaeroides RC yakoreshejwe nk’akarorero (ID ya PDB: 5LSE) (46). Koresha igikoresho ca “ikarata ibereye” muri UCSF Chimera kugira ngo uhuze n’ikigereranyo cakozwe n’ikarata (47), utere imbere mu miterere ya poroteyine, n’ibindi bifasha [4×BChl a (izina ry’ibisigazwa vy’ibitabo vy’ibinyabuzima = BCL), 2×BPh a (BPH), ubwoko bumwe canke bubiri bwa UQ10 (U10), bumwe butari he-heron na 3,4-dihidroheksakarbonilkolini (QAK)] ukoreshe Coot (48) kugira ngo wongereko. Kubera ko QAK itaboneka mu bubiko bw’ibitabu vy’ibintu bimwe bimwe, yarashizweho hakoreshejwe igikoresho ca eLBOW kiri muri PHENIX (49).
Inyuma y’aho, haciye hubakwa igice ca LH1. Mu ntango, igikoresho co kwubaka cikora kiri muri PHENIX (49) carakoreshejwe mu kwubaka igice c’urutonde rwa LH1 hakoreshejwe ikarita n’urutonde rwa poroteyine LH1-α na LH1-β nk’inyungu. Hitamwo igice ca LH1 cuzuye kuruta ibindi vyose, ugikuremwo maze ugishire muri Coot, wongeremwo n’amaboko urutonde rubuze muri co, hanyuma ushiremwo n’amaboko urutonde rwose imbere yo kwongerako BCls zibiri a (BCL) na spirilloxanthin (CRT) [bishingiye ku Rps zijanye n’ivyo Uburemere bw’ibintu birimwo caroten n’ivyo bizwi. Ubwoko (17)]. Kopa igice ca LH1 cose, hanyuma ukoreshe “Igikoresho co Gushiramwo Ikarata” ca UCSF Chimera kugira ngo ushire ahantu hafi y’aho hatari akarorero k’uburemere bwa LH1, hanyuma ugitunganye muri Coot; subiramwo iyo nzira gushika ibice vyose vya LH1 bigereranywa. Ku bijanye n’imiterere ya RC-LH114-W, mu gukuraho uburemere butagenewe muri Coot, poroteyine iragabanywa mu bice bisigaye bitagira poroteyine biri ku ikarita ya USCF Chimera maze igikoresho ca Autobuild kigakoreshwa mu gushinga urugero rwa mbere, n’ibice bisigaye (proteine-W) Modeling. Mu gitabu ca FENISI (49). Yongerako urutonde rwose rubuze ku citegererezo kivuye muri Coot (48), hanyuma utunganye n’amaboko igice cose. Ivyo bihimba vy’umubiri bisigaye bitagabanijwe bihuye n’uguhuza amavuta (ID y’ibitabo vy’ibinyabuzima bimwe bimwe vya PDB vya CDL = CDL, POPC = 6PL na POPG = PGT), igikoresho co gusukura β-DDM (LMT) n’ibihimba vy’umubiri bita UQ10 (U10). Koresha PHENIX optimization (49) n’ugutunganya kw’amaboko muri Coot (48) kugira ngo utunganye neza igishushanyo c’intango cuzuye gushika imibare y’igishushanyo n’uburyo bwo kubona bw’ivyo bihuye bidashobora kurushiriza gutera imbere. Ubwa nyuma, koresha LocScale (50) kugira ngo ushiremwo ikarita y’aho hantu, hanyuma ukore izindi nzira nyinshi zo gushushanya uburemere butagenewe be n’ugutuma ibintu bigenda neza mu buryo bwikora no mu buryo bw’amaboko.
Ivyo bihimba vy’umubiri, ivyo bihimba vy’umubiri n’ibindi bihimba vy’umubiri bifise amavuta n’ibindi bihimba vy’umubiri bifatanye n’uburemere bwavyo vyerekanywe ku bishushanyo 1 na 2. S18 gushika kuri S23. Amakuru y'imibare y'akarorero ka nyuma yerekanwa mu ndome S1.
Keretse iyo bivuzwe ukundi, imishwarara y’ugufata UV/Vis/NIR yakoranijwe ku gipimo c’imishwarara Cary60 (Agilent, USA) ku bihe vy’imirongo 1 kuva kuri 250 nm gushika kuri 1000 nm n’igihe co gukorana c’amasegonda 0.1.
Ivyo bipimo bishire mu cuvette ya quartz ifise inzira ya mm 2 ija kuri A880 ya 1, hanyuma ushire hamwe umuco w’ugufata hagati ya 400 na 1000 nm. Ivyo bimenyetso vy’uruziga vy’imirongo ibiri vyakoranijwe ku gikoresho co gupima ibara ry’umuco Jasco 810 (Jasco, Ubuyapani) ku 1 nm hagati ya 400 nm na 950 nm ku rugero rwo gupima 20 nm min-1.
Igitigiri c’uguzimangana kw’ibihimba vy’umubiri kimenyekana mu gucapura igice c’umubiri gifise A880 c’amabara nka 50. Gucapura umubare w’amazi 10μl mu mazi 990μl y’ubufatanye canke methanol, hanyuma ushire hamwe umuco w’ugufata ubwo nyene kugira ngo ugabanye ukuntu BChl ishobora gusenyuka. Ivyo BChl biri muri buri nkuru ya methanol vyaharuwe n’igipimo co kuzimangana ku 771 nm ca 54,8 mM-1 cm-1, hanyuma igipimo co kuzimangana kiramenyekana (51). Gabanya ivyiyumviro vya BChl vyagereranywa na 32 (RC-LH114-W) canke 36 (RC-LH116) kugira ngo umenye ivyiyumviro vy’ivyuma bikomeye, ivyo bikaba bikoreshwa mu kumenya umurongo w’ugunywa kw’ico kigereranyo nyene cakoranijwe mu gipimo c’uguzimangana kw’ivyuma. uruhande rumwe. Hafashwe ingero zitatu zisubiwemwo ku citegererezo kimwekimwe cose, kandi umubare w’ugufata amazi w’agaciro ka BChl Qy warakoreshejwe mu kubara. Igitigiri c’ukuzimangana kwa RC-LH114-W gipimwa ku 878 nm ni 3280±140 mM-1 cm-1, mu gihe igitigiri c’ukuzimangana kwa RC-LH116 gipimwa ku 880 nm ni 3800±30 mM-1 cm-1.
UQ10 yagereranywa hakurikijwe uburyo buri muri (52). Muri make, HPLC y’inzira y’inyuma (RP-HPLC) yakozwe hakoreshejwe ubuhinga bwa Agilent 1200 HPLC. Ushire nk’ibice 0,02 nmol vya RC-LH116 canke RC-LH114-W mu 50μl y’ivyuma bita methanol:kloroforme 50:50 birimwo 0,02% (w/v) vy’ivyuma vy’ivyuma, hanyuma ushiremwo ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma. 40°C mu gikoresho co gusukura HPLC (80:20 metanoli:2-propanol) ku nkingi ya ×25 cm. Gukora igikorwa co gukuraho isocratique mu gikoresho co gusukura HPLC kugira ngo ugenzure ukuntu amazi afata amazi ku 275 nm (UQ10), 450 nm (carotenoïdes) na 780 nm (BChl) mu kiringo c’isaha 1. Isonga ryo mu 275 nm chromatogram ku minota 25,5 ryari rishizwemwo, ritagira ibindi bihimba bishobora kumenyekana. Uburebure buhurikiyemwo bukoreshwa mu kubara umubare w’amamolare ya UQ10 yakuwemwo hakurikijwe umurongo w’ugupima waharuwe uhereye ku gutera urushinge rw’ibipimo vyiza kuva kuri 0 gushika kuri 5,8 nmol (Ishusho S14). Icogereranyo cose carasuzumwe mu bisubizo bitatu, kandi ikosa ryavuzwe rihuye n’i SD y’umubare w’abantu.
Igisubizo kirimwo umugwi wa RC-LH1 gifise ubushobozi bwo gufata Qy burengeye 0,1 carateguwe hakoreshejwe 30 μM y’umutima w’ifarashi ugabanutse c2 (Merck, UK) na 0 gushika kuri 50 μMUQ2 (Merck, UK). Ivyitegererezo bitatu vy’imililitiro 1 vyateguwe ku rwego rwose rwa UQ2 hanyuma bishirwa mu mwiza ijoro ryose ku bushuhe bwa 4°C kugira ngo bimenyere neza umwiza imbere y’uko bipimwa. Ico gisubizo cashizwe mu gikoresho co gupima umuco w’izuba OLIS RSM1000 gifise urubeya rw’umurongo w’uburebure bwa 300 nm/500, 1,24 mm y’inzira y’inzira, 0,12 mm hagati n’inzira y’inzira y’amazi y’inzira y’amazi 0,6 mm. Igicuncuro c’uburebure bwa 600 nm gishirwa ku bwinjiriro bw’ibarabara ry’amafoto ry’akarorero n’ibarabara ry’ibarabara ry’ibarabara ry’ibarabara kugira ngo umuco w’ugukangura ntubeho. Ivyo bimenyetso vyakurikiranywe ku 550 nm n’igihe co gukorana c’amasegonda 0,15. Umuco w’ugukangura usohoka ku nzira y’umuco w’uburemere bwa 880 nm M880F2 (Thorlabs Ltd., Ubwongereza) biciye ku nsinga y’umuco ku 90% biciye ku gikoresho co kugenzura DC2200 (Thorlabs Ltd., Ubwongereza) kandi usohoka ku nzira y’umuco ku mfuruka ya°0. Ico gikoresho co gupima gihanganye n’indorerwamo kugira ngo kigarure umuco uwo ari wo wose utari waranywewe n’ico kigereranyo mu ntango. Gukurikirana ukuntu umuco unyura 10 s imbere y’uko umuco ushika 50 s. Hanyuma, iyo nzira y’ugufata amazi yarasuzumwe mu kiringo c’amasegonda 60 mu mwiza kugira ngo hasuzumwe urugero quinolol igabanyako cytochrome c23 + (raba igishushanyo S8 ku makuru atagiramwo ivyatsi).
Amakuru yatunganijwe mu gushiramwo igipimo c’intango c’umurongo mu masegonda 0,5 gushika ku 10 (bivanye n’urugero rwa UQ2) no gupima urugero rw’ibipimo vyose bitatu ku rugero rwose rwa UQ2. Igitigiri ca RC-LH1 caharuwe n’igipimo c’uguzimangana cakoreshejwe mu guhindura igipimo mu bushobozi bw’ivyuma bitera umuriro, gishushanijwe muri Origin Pro 2019 (OriginLab, USA), kandi gihuye n’akarorero ka Michaelis-Menten kugira ngo hamenyekane agaciro ka Km na Kcat.
Ku bijanye n’ingero z’ugufata amazi mu gihe gito, igipimo ca RC-LH1 caraciwemwo ~2μM mu buffer ya IMAC irimwo 50 mM y’umunyu w’amazi (Merck, USA) na 0.4 mM ya Terbutin (Merck, USA). Asidi ascorbique ikoreshwa nk’umutanzi w’ama elegitoroni y’ikimazi, tert-butaclofen na yo ikoreshwa nk’inzitizi ya QB kugira ngo umutanzi nyamukuru wa RC agume agabanutse (ni ukuvuga ko atagira photooxidation) mu gihe cose c’ugupima. Nk’imililitiro 3 z’ivyafashwe vyongerwa ku gice gizunguruka gisanzwe (nk’ibilometero 0,1 mu gipimo, 350 RPM) gifise uburebure bw’inzira y’amaso y’iminota 2 kugira ngo ivyerekanwa biri mu nzira ya laser bibe bifise umwanya uhagije wo guhindura umwiza hagati y’impumuro z’ugukangura. Koresha ~100-fs ivyuma vy’umuco kugira ngo ukomeze ubuhinga bwa laser bwa Ti: Sapphire (Spectra Physics, USA) kugira ngo ushire umunezero ku 880 nm ku rugero rw’ugusubiramwo rwa 1 kHz (20 nJ ku NIR canke 100 nJ ku Vis). Imbere yo gukusanya amakuru, ushire iyo nkuru mu rumuri rw’ugukangura mu minota nka 30. Gushirwa ahabona bizotuma QA idakora (bishoboka ko QA igabanywa rimwe canke kabiri). Ariko ndabinginze mumenye ko iyo nzira ishobora gusubirwamwo kuko inyuma y’igihe kirekire c’ukumenyera umwiza, RC izosubira buhoro buhoro mu gikorwa ca QA. Igikoresho co gupima ibara ry'umuco (Ultrafast Systems, USA) carakoreshejwe mu gupima ibara ry'umuco ry'igihe gito rifise igihe co guteba co kuva kuri -10 gushika kuri 7000 ps . Koresha porogarama ya Surfast Xplorer (Ultrafast Systems, Leta Zunze Ubumwe za Amerika) kugira ngo ukureho amakuru, hanyuma uyashire hamwe maze uyashire mu rugero rumwe. Koresha porogarama ya CarpetView (Light Conversion Ltd., Lithuania) kugira ngo ukoreshe amakuru yose hamwe kugira ngo ubone imirongo itandukanye ijyanye n’ukubora, canke ukoreshe igikorwa gifatanya ibimenyetso vyinshi n’inyishu y’igikoresho kugira ngo bihure n’iterambere ry’imirongo y’uburebure bw’umuvuduko umwe mu OriginLa (OriginLa).
Nk’uko twabibonye haruguru (53), hateguwe filime y’ubuhinga bw’izuba irimwo LH1 complex idafise antene ya RC n’iya LH2 y’inyuma. Ico gipfukisho carashizwe mu mazi 20 mM tris (pH 8.0) hanyuma gishirwa mu cuvette y’amabuye y’agaciro yitwa quartz ifise inzira y’amaso y’iminota 2. Igikoresho ca laser c’uburemere bwa 30nJ carakoreshejwe kugira ngo gitere umunezero ku 540 nm n’igihe co gutevya co kuva kuri -10 gushika kuri 7000 ps. Gukora amakuru nk’uko vyavuzwe ku Rps. pal akarorero.
Iryo banga ryaciwe mu bipande biciye mu gucapura ku 150.000 RCF mu kiringo c’amasaha 2 ku 4°C, hanyuma ubushobozi bwaryo bwo gufata ku 880 nm burasubirwamwo mu 20 mM tris-HCl (pH 8.0) na 200 mM NaCl. Ushiremwo uruzitiro mu gutera buhoro buhoro mu 2% (w/v) β-DDM mu kiringo c’isaha 1 mu mwiza ku 4°C. Ico kigereranyo caciwe mu mazi y’ibara ry’agahama (pH 8,0) (TEAB; Merck, UK) ku rugero rwa 2,5 mg ml-1 (isesengura rya Bio-Rad). Ibindi bikorwa vyakozwe bivuye ku buryo bwasohowe mbere (54), hatangurwa no guhindura 50 μg ya poroteyine mu 50 μl ya TEAB yose hamwe irimwo 1% (w/v) ya sodium laurate (Merck, UK). Inyuma y’ugutera ijwi ry’umuriro mu kiringo c’amasegonda 60, vyagabanutse n’imiti 5 mM ya tris (2-carboxyethyl) phosphine (Merck, Ubwongereza) ku 37°C mu minota 30. Ku bijanye n’ugukora S-alkylation, ushiremwo ivyerekanwa n’amazi y’ivyatsi 10 mM (Merck, UK) hanyuma uyishiremwo mu mazi y’isopropanol 200 mM mu minota 10 ku bushuhe bwo mu cumba. Ivyo bihimba vy’umubiri vyakozwe mu kwongerako 2 μg y’umuvango wa trypsine/endoproteinase Lys-C (Promega UK) hanyuma bishirwa ku bushuhe bwa 37°C amasaha 3. Ivyo bihimba vy’umubiri vyakuwe mu kwongerako 50 μl y’ivyatsi vy’ivyatsi vy’ivyatsi vy’ivyatsi vy’ivyatsi vy’ivyatsi vy’ivyatsi (TFA; Thermo Fisher Scientific, UK) hanyuma bikavyuka mu kiringo c’amasegonda 60. Ugutandukanya inzira kwatewe imbere n’ugucapura ku 15.700 RCF mu minota 5. Dukurikije amategeko y’umuhinguzi, hakoreshejwe inkingi y’ivyuma yitwa C18 (Thermo Fisher Scientific, UK) kugira ngo umuntu ashobore gufukura neza no gukura umunyu mu gice co hasi kirimwo iyo peptide. Inyuma yo kwuma hakoreshejwe ubuhinga bwo gupima ubusa, iyo nkuru yashizwe mu 0,5% ya TFA na 3% ya acetonitrile, hanyuma 500 ng zisuzumwa hakoreshejwe ubuhinga bwa nanoflow RP chromatography bufatanijwe n’ubuhinga bwa mass spectrometry hakoreshejwe ibipimo vy’urutonde vyavuzwe mu buryo burambuye mbere.
Koresha MaxQuant v.1.5.3.30 (56) ku kumenya poroteyine n’ingero kugira uronde Rps. urutonde rw’ibinyabuzima vy’ibinyabuzima bita palustris (www. Amakuru y'ubuhinga bwo gupima uburemere bw'ibinyabuzima yashizwe mu gisata c'ububiko bw'ibinyabuzima biciye mu bubiko bw'abafatanyabikorwa ba PRIDE (http://proteomecentral.proteomexchange.org) munsi y'ikimenyetso c'amakuru PXD020402.
Kugira ngo hakorwe isesengura n’ubuhinga bwa RPLC bufatanijwe n’ubuhinga bwo gupima uburemere bw’ibintu bikoreshwa n’umuyagankuba, RC-LH1 complex yateguwe ikoresheje Rps z’ubwoko bw’ishamba. Hakoreshejwe uburyo bwasohowe mbere (16), ivyiyumviro vya poroteyine vyavuye mu ngingo z’ibimera vy’ibitoke vyari 2 mg ml-1 mu Hepes 20 mM (pH 7,8), NaCl 100 mM na 0,03% (w/v) β- (isesengura rya Bio-Rad) ) DDM. Dushingiye ku mategeko y’umuhinguzi, ukoreshe igikoresho co gusukura 2D (GE Healthcare, USA) kugira ngo ukuremwo 10 μg ya poroteyine mu buryo bwo gutera imvura, hanyuma uyishire mu 20 μl y’aside formique (FA) 60% (v / v), Acetonitrile 20% (v / v) n’amazi 20% (v/)vter. Mikorolitiro zitanu zasuzumwe n’ubuhinga bwa RPLC (Dionex RSLC) bufatanijwe n’ubuhinga bwo gupima uburemere bw’ibintu (Maxis UHR-TOF, Bruker). Koresha inkingi ya MabPac 1,2×100 mm (Thermo Fisher Scientific, Ubwongereza) ku gutandukanya ku 60°C na 100μlmin -1, n’urugero rwa 85% (v / v) umunyu A [0,1% (v / v) FA na 0,02% (V/v) umuti B5/v a [0.1%(v/v) FA na 0.02%(v/v) muri 90%(v/v) acetonitrile TFA] Hakoreshejwe isoko y’iyonization y’umuyagankuba n’ibipimo vy’imbere mu minota irenga 60, igipimo c’uburemere bw’ibintu kironka 100 gushika kuri 2750 m/z (igipimo c’uburemere n’uburemere). Ufashijwe n’igikoresho ca FindPept (https://web.expasy.org/findpept/), ushireho ikarita y’urugero rw’uburemere bw’ibintu ku bice vy’ivyo bihimba.
Utugingo ngengabuzima twakuze amasaha 72 munsi y’umuco w’imiliyoni 100 NF-muto (10μMm-2 s-1), hagati (30μMm-2 s-1) canke munini (300μMm-2 s-1). M22 (M22 aho hakurwamwo sulfate y’amoniyumu, succinate ya sodium igasubirizwa n’acetate ya sodium) mu cupa ry’imiliyoni 100 ry’ivyuma (23). Mu nzinguzingu zitanu z’amasegonda 30, amabuye y’ibirahuri y’imikoro 0,1 yarashizweko amabuye ku rugero rw’uburemere bwa 1:1 kugira ngo ahindure ingingo ngenderwako maze akonjere ku barafu iminota 5. Ivyo bintu bidashobora gusenyuka, utugingo ngengabuzima tutavunitse n’ibipande vy’ibirahuri vyakuweho biciye mu gucapura ku 16.000 RCF mu minota 10 mu gikoresho co gucapura ku ntebe. Iryo banga ryatandukanijwe mu gikoresho citwa Ti 70.1 gifise RCF 100.000 mu tris-HCl 20 mM (pH 8.0) gifise 40/15% (w/w) y’isukari mu masaha 10.
Nk’uko vyavuzwe mu gikorwa cacu c’imbere, immunodetection y’ikimenyetso ciwe kuri PufW (16). Muri make, igice c’imbere gisukuye (11,8 nM) canke uruzitiro rurimwo ivyiyumviro bimwe vy’i RC (bigenwa n’ugukuraho ibara ry’itandukaniro ryagabanutse no guhuza umuzigo uri ku gitereko c’amabara) mu 2x SDS loading buffer (Merck, UK) yahinduwe incuro zibiri. Ivyo bihimba vy’umubiri vyatandukanijwe ku gikoresho co gupima 12% bis-tris NuPage gel (Thermo Fisher Scientific, mu Bwongereza). Igikoresho ca gel carashizweko ubururu bukeye bwa Coomassie (Bio-Rad, UK) kugira ngo gishiremwo no kubona mu ciyumviro igice ca RC-L. Ivyo bihimba vy’umubiri biri kuri iyo gel ya kabiri vyashizwe mu gipfukisho c’umubiri (Thermo Fisher Scientific, UK) kugira ngo bikoreshwe mu gupima ubudahangarwa bw’umubiri. Igipande ca PVDF carazibiwe mu 50 mM tris-HCl (pH 7,6), 150 mM NaCl, 0,2% (v / v) Tween-20 na 5% (w / v) c’amata y’ifu, hanyuma gica giterwa n’umubiri w’intango urwanya His (mu buffer mH5p 7,6), 150 mM NaCl na 0,05% (v/v) Tween-20] mu 1:1000 A190-114A, mu bigo vy’ubushakashatsi vya Bethyl, muri Leta Zunze Ubumwe za Amerika) mu masaha 4. Inyuma yo kwozwa incuro 3 mu minota 5 mu mazi y’imiti irwanya indwara, iyo nzira yashizwe hamwe n’umuti w’imbeba (Sigma-Aldrich, UK) (wahinduwe 1:10.000 mu mazi y’imiti irwanya indwara) Wincubate kugira ngo bishobore kumenya (iminota 5 inyuma yo kwozwa 3 mu mazi y’imiti irwanya indwara E ESTAR0) (Cyanagen, mu Butaliyano) n’ivy’amashusho y’i Amersham 600 (Ivy’ubuvuzi bwa GE, mu Bwongereza).
Mu gushushanya ugusangira kw’uburemere bw’umurongo w’ibara canke immunoassay, gushiramwo ahantu hari munsi y’isonga no kubara igipimo c’uburemere bwa RC-L (ibara ry’umubiri) na Protein-W (immunoassay), mu ImageJ (57) Gukora ishusho. Ivyo bipimo vyahinduwe mu bipimo vya molar mu kwiyumvira ko igipimo ca RC-L na proteine-W mu citegererezo ca RC-LH114-W gisukuye cari 1:1 no gusanura amakuru yose nk’uko bibereye.
Ku bijanye n’ibikoresho vy’inyongera vy’iyi ngingo, raba kuri http://advances.sciencemag.org/cgi/ibirimwo/vyuzuye/7/3/eabe2631/DC1.
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Igishushanyo c’umutego w’umuco 1 gikomeye kiri mu kibanza c’ugusubiza kiratanga ubumenyi bushasha ku bijanye n’ingene quinone ikora.
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Igishushanyo c’umutego w’umuco 1 gikomeye kiri mu kibanza c’ugusubiza kiratanga ubumenyi bushasha ku bijanye n’ingene quinone ikora.
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