Kero yazvino: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Chikamu chekugadzirisa chiedza che 1 (RC-LH1) ndicho chikamu chikuru che photosynthetic che purple phototrophic bacteria. Takaunza maumbirwo maviri e cryo-electron microscopy e RC-LH1 complex kubva ku Rhodopseudomonas palustris. Maumbirwo e 2.65-Å resolution ye RC-LH114-W complex ane 14 subunit LH1 loops akakomberedza RC, ayo anokanganiswa ne protein W, nepo complex isina protein-W iri RC composition yakakomberedzwa ne RC. Yakavhara 16 subunit LH1 loop. Kuenzanisa kwezvimiro izvi kunopa ruzivo pamusoro pesimba re quinone mu RC-LH1 complex, kusanganisira shanduko dzisina kutsanangurwa dze conformational pakusunga quinone panzvimbo ye RC QB, pamwe nenzvimbo ye assistant quinone binding sites, izvo zvinobatsira Kuaendesa ku RC. Maumbirwo akasiyana e W protein anodzivirira kuvharwa kwe LH1 loop, nokudaro achigadzira nzira yekukurumidzisa quinone/quinolone exchange.
Simba rinopihwa ne photosynthesis rinogona kuchengetedza hupenyu hwese panyika, uye rine mukana mukuru wekugadzira solar biotechnology. Kunyange zvazvo richikurudzira photosynthesis yepasi rose, mabhakitiriya epepuru ephototrophic anoratidzawo nzira dzakasiyana dzesimba uye kugona kwe metabolic. Anogona kudzivirira photosynthesis uye kukura sezvo mabhakitiriya eheterotrophic murima, anogona kugadzirisa nitrogen necarbon dioxide, kugadzira hydrogen, uye kushatisa aromatic compounds (1-3). Kuti ape simba remaitiro aya, chiedza chinofanira kushandurwa nekukurumidza uye zvinobudirira kuita simba remakemikari. Maitiro aya anotanga apo light-trapping antenna complex inotora chiedza uye inoendesa simba rakabatirirwa kunzvimbo ye reaction (RC), nokudaro ichitanga kupatsanurwa kwechaji (4 - 7). Chikamu chekutanga che photosynthesis mu purple phototrophic bacteria chinoumbwa ne type 2 RC, yakakomberedzwa ne light-harvesting complex 1 (LH1), ichiumba RC-LH1 core complex. LH1 inoumbwa ne array ye curved αβ heterodimers, imwe neimwe inosunga mabacteria maviri chlorophyll (BChl) mamorekuru uye carotenoids imwe kana mbiri (8-12). Antenna yeLH1 iri nyore ine 16 kana 17 αβ heterodimers dzakakomberedza RC (9-13) mu closed loop, asi mune dzimwe core complexes, transmembrane peptides inodzivisa kuenderera mberi kweLH1 yakapoteredza, Izvi zvichikurudzira kupararira kwequinol/quinone pakati peRC ne cytochrome bc1 complex (11, 13-15). Chirimwa chepepuru chephototrophic Rhodopseudomonas (Rps.) imhando yechinhu chinogona kunzwisisa simba uye electron transfer inotsigira photosynthesis. Chimiro chekutanga chekristaro cheRps. Muenzaniso wepalustris RC-LH1 complex iRC, yakakomberedzwa ne15 heterodimeric LH1 loops, iyo inokanganiswa neprotein isingazivikanwe inonzi "Protein W" (14). Protein-W yakazozivikanwa seRPA4402, inova protein ye10.5kDa isina hunhu ine helices nhatu dzakafanotaurwa dze transmembrane (TMH) (16). Tinokurudzira kutumidza zita idzva remagene e-rpa4402 anoburitsa protein W kuita pufW kuti rienderane nemazita anoshandiswa kumagene anoburitsa RC-L, M (pufL, pufM) uye LH1α, β (pufA, pufB) subunits. Zvinofadza kuti, protein-W inowanikwa chete muinenge 10% yeRC-LH1, zvichiratidza kuti Rps. palustris inogadzira ma complexes maviri akasiyana eRC-LH1. Pano, tinotaura nezve high-resolution cryo-EM (cryo-EM) structures ema core complexes maviri, rimwe rine protein W uye 14 αβ heterodimers, rimwe risina protein W uye closed 16 Heterodimer LH1 loop. Maumbirwo edu anomiririra shanduko huru mukunzwisisa RC-LH1 complex yeRps. palustris, nekuti takaongorora huwandu hwemhando imwe neimwe uye tine resolution yakakwana yekupa zvakajeka peptide yega yega uye bound pigments uye related lipids uye quinones. Kuenzanisa kwezvimiro izvi kunoratidza kuti mapuroteni matatu eTMH-W asina kuwanikwa mune imwe RC-LH1 complex kusvika pari zvino anogadzira quinone channel yekukurumidzisa kuchinjana kwequinone/quinolone. Nzvimbo dzakawanda dzakachengetwa dzekubatanidza lipid nequinone dzakaonekwa, uye takaratidza shanduko itsva yekuchinja mushure mekusanganiswa kwequinone neRC, iyo inogona kukodzera photosystem II (PSII) RC yezvipenyu zvine oxygen. Zvatakawana zvinopa ruzivo rutsva nezve kinetics yekubatanidza kwequinone/quinolone uye kuchinjana muRC-LH1 core complex yepurple phototrophic bacteria.
Kuti tikwanise kuita kuti ongororo yakadzama yemacomplex maviri anowanikwa muRps. palustris ive nyore, tinoparadzanisa RC-LH1 yega yega nenzira dzebiochemical. Protein W-deficient complex (inozonzi ΔpufW) yakacheneswa kubva mustrain isina pufW gene (16), uye RC-LH1 complex imwe chete ndiyo inogona kugadzirwa. Protein W-containing complex inogadzirwa nestrain. Protein W yestrain iyi inogadziriswa ne10x His tag paC-terminus yayo, kuitira kuti protein W-containing complex ikwanise kubatanidzwa zvinobudirira neprotein W yakawanda isina nekudzivirira simbi. Complex iyi inoparadzaniswa zvinobudirira (16) Affinity Chromatography (IMAC).
Sezvakaratidzwa paMufananidzo 1, macomplex ese ari maviri ane sub-unit RC nhatu (RC-L, RC-M uye RC-H) dzakakomberedzwa neLH1 antenna. Chimiro che2.80-A checomplex isina protein-W chinoratidza 16 αβ heterodimers, zvichigadzira loop yakavharwa yeLH1 yakakomberedza RC, pano ichizonzi RC-LH116 complex. Chimiro che2.65Å che protein-W-containing complex chine 14-heterodimer LH1 yakakanganiswa neprotein-W, pano ichizonzi RC-LH114-W.
(A naB) Chimiro chepamusoro chemusanganiswa. (C naD) Mapigmenti akabatana anoratidzwa mumatanda. (E naF) Macomplex anoonekwa kubva pamusoro pe cytoplasm ane ma peptides neLH1 subunits anomiririrwa mumakatuni, uye anoverengerwa newachi kubva pa protein-W gap [inoenderana neRba numbering. sphaeroides complex (13)]. Kune LH1-α, ruvara rwe protein subunit yeyero; kune LH1-β, ruvara rwe protein subunit ibhuruu; kune protein-W, protein itsvuku; kune RC-H, i cyan; kune RC-L, iOrange; kune RC-M, magenta. MaCofactors anomiririrwa nematanda, girini inomiririra BChl uye BPh mamorekuru, purple inomiririra carotenoids, uye yero inomiririra mamorekuru eUQ10. (G naH) Maonero akakwidziridzwa e protein-W gap munzvimbo yakaenzana yeRC-LH114-W complex (G) uye RC-LH116 complex (H). MaCofactor anoratidzwa muchimiro chekuzadza nzvimbo, chelated quinone inoratidzwa mubhuruu. Mukana weprotein-W unoonekwa nemutsetse webhuruu wakachekwa mu (G), uye maburi madiki apo quinone/quinolol inopararira paLH116 ring anoonekwa nemutsetse mutema wakachekwa mu (H).
Mufananidzo 1 (A naB) unoratidza RC yakakomberedzwa neLH1αβ heterodimers dzakavhurika kana dzakavharika, imwe neimwe ichisunga maBChl maviri uye carotenoid imwe (Mufananidzo 1, C naD). Zvidzidzo zvekare zvakaratidza kuti Rps ndiyo LH1 complex. Munzira yekugadzira spirulina xanthin, mhando idzi dzine huwandu hwakasanganiswa hwema carotenoids (17). Zvisinei, spiropyrroxanthin ndiyo carotenoid inotonga uye huwandu hwayo hunogutsa. Saka, takasarudza kutevedzera spiroxanthin panzvimbo dzese dzeLH1 dzinosunga. Ma alpha ne beta polypeptides iTMH imwe chete ine nzvimbo pfupi dzekunze kwemembrane (Mufananidzo 1, A, B, E, naF). Kunyangwe huwandu hwema residues gumi nemanomwe paC-terminus husina kuonekwa, alpha polypeptide yakakamurwa kubva kuMet1 kuenda kuAla46 muma complexes ese ari maviri. β polypeptide yakaderedzwa kubva kuGly4 kuenda kuTyr52 muRC-LH116, uye kubva kuSer5 kuenda kuTyr52 muRC-LH114-W. Hapana huwandu hwe3 kana 4 N-terminal kana 13 C-terminal residues hwakaonekwa (Mufananidzo S1). Kuongororwa kweMass spectrometry ye mixed RC-LH1 complex yakagadzirwa kubva ku wild-type strain kwakaratidza kuti nzvimbo isipo yaive mhedzisiro ye heterologous cleavage yeaya ma peptides (Mufananidzo S1 naS2). N-terminal formylation ye α-Met1 yakaonekwawo (f). Kuongororwa kwakaratidza kuti α-peptide ine residues fMet1 kuenda kuAsp42/Ala46/Ala47/Ala50, uye β-peptide ine residues Ser2 kuenda kuAla53, iyo inoenderana zvakanaka ne low-temperature EM density map.
Kubatana kweα-His29 neβ-His36 kunoita kuti BChls itarisane; αβ heterodimer yega yega inoungana nevavakidzani vayo kuti igadzire loop yakavhurika (RC-LH114-W) kana loop yakavharika (RC-LH116) yakatenderedza RC. Iyo exciton coupled pigment array (Mufananidzo 1, C naD). Zvichienzaniswa ne 877 nm band ye RC-LH114-W, iyo 880 nm absorption red shift ye RC-LH116 i 3 nm (Mufananidzo 2A). Zvisinei, circular dichroism spectrum inenge yakafanana (Mufananidzo 2B), zvichiratidza kuti kunyangwe paine musiyano wakajeka pakati pe loops dzakavhurika nedzakavharwa, nharaunda yeBChls yemuno yakafanana zvikuru. Iyo absorption redshift inogona kunge iri mhedzisiro yekudzikira kwekufamba kwekupisa uye kuwedzera kugadzikana pa loop yakavharika (18, 19), shanduko mukubatana kwe pigment inokonzerwa ne closed loop (20, 21), kana musanganiswa wemhedzisiro iyi miviri (11).
(A) Ultraviolet/visible/near-infrared absorption spectrum, peaks dzacho dzakanyorwa ne pigments dzadzo dzinoenderana uye dzakagadziriswa kusvika paBPh peak pa775 nm. (B) Circular dichroism spectrum yakajairwa kusvika paBChl absorbance pa805 nm. (C naD) ΔA spectra yakasarudzwa kubva ku RC-LH114-W complex (C) uye RC-LH116 complex (D). Kuti zvienzaniswe zviri nani, spectra dzese dzakajairwa kusvika pa ∆A ye −A pa 0.2 ps. (E) Mwero we cytochrome c2 oxidation mushure mekuvhenekerwa nekuvapo kwehuwandu hwakasiyana hweUQ2 (ona Mufananidzo S8 kuti uwane data raw). (F) Mumasero akakurira pasi pechiedza chakaderera, chepakati kana chakakwirira (10, 30 kana 300μMm-2 s-1, zvichiteerana), protein W neRC-L subunits mu purified complex uye separated membrane ratio. Sarudza huwandu hweprotein uchishandisa SDS-polyacrylamide gel electrophoresis uye immunoassay (ona Mufananidzo S9 kuti uwane data raw). Sarudza huwandu hunoenderana ne RC-LH114-W complex yakacheneswa. Chiyero che stoichiometric cheRC-L kune protein-W ye complex i1:1.
MaBChl ari panzvimbo 1 mu deformed αβ14 loop yeRC-LH114-W (Mufananidzo 1, A, C, na E) ari pedyo neRC primary donor (P) ne6.8Å kupfuura maBChl akaenzana muRC-LH116 (Mufananidzo 1, B, D, na F, naMufananidzo S3); zvisinei, transient absorption kinetics yema complexes maviri anoratidza kuti kune RC-LH114-W naRC-LH116, excitation energy transfer time constants kubva kuLH1 kuenda kuRC i40 ±4 na44±3 ps (Mufananidzo 2). , C naD, Mufananidzo S4 naTafura S2). Hapana musiyano wakakosha mukufambiswa kwemagetsi mukati meRC (Mufananidzo S5 nemagwaro ekuwedzera ane chekuita nazvo). Tinofungidzira kuti close correspondence yenguva yekufambiswa kwesimba pakati peLH1 naRC-P imhaka yedaro rakafanana, angle uye potential energy yeBChl yakawanda muLH1 loops mbiri. Zvinoita sekuti kuongorora patani yesimba reLH1 kuti usvike kure zvishoma hakusi kukurumidza kupfuura kutamiswa kwesimba zvakananga kubva panzvimbo dzisina kunaka kuenda kuRC. Kutenderera kweLH1 kwakavhurika muRC-LH114-W kunogonawo kufamba zvishoma nekupisa pasi pemamiriro ekupisa akaderera ekuongorora chimiro, uye kune αβ14 ring conformation yakareba patembiricha yemumba kubva kure kwepigmentation yeβBChls panzvimbo yeRC 1.
Iyo RC-LH116 complex ine 32 BChls uye 16 carotenoids, uye kurongeka kwayo kwakafanana nekwakawanikwa kubva kuThermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 strain (PDB ID 7C9R) (12) uye green algae (Blc.viridis) (PDB ID 6ET5) (10). Mushure mekuenderana, kutsauka kudiki chete munzvimbo dze αβ heterodimers kwakaonekwa, kunyanya 1-5, 15, uye 16 (Mufananidzo S6). Kuvapo kweprotein-W kune simba guru pachimiro cheLH1. MaTMH ayo matatu akabatana nezvidimbu zvipfupi, neN-terminal iri kurutivi rwe lumen rwe complex uye C-terminal iri kurutivi rwe cytoplasmic (Mufananidzo 1A ne 3, A kusvika D). Mapuroteni-W anonyanya kuvhiringidzika ne hydrophobic (Mufananidzo 3B), uye TMH2 neTMH3 zvinosangana neLH1αβ-14 kuti zvigadzire pamusoro pe transmembrane (Mufananidzo 3, B na E kusvika G). Nzvimbo iyi inonyanya kuumbwa nePhe, Leu naVal residues munzvimbo ye transmembrane. Aya ma residues akafukidzwa ne hydrophobic amino acids uye αβ-14 pigments. Mamwe ma polar residues anobatsirawo mukubatana, kusanganisira hydrogen bond pakati peW-Thr68 na β-Trp42 pamusoro pe complex cavity (Mufananidzo 3, F na G). Pamusoro pe cytoplasm, Gln34 iri padyo ne keto group ye αβ-14 carotenoids. Pamusoro pezvo, n-dodecyl β-d-maltoside (β-DDM) molecule yakagadziriswa, uye hydrophobic tail yayo yakatambanudzwa kusvika pakubatana pakati pe protein-W na αβ-14, uye lipid tail inogona kunge iri mumuviri. Takaonawo kuti nzvimbo dzeC-terminal resolution dzeprotein W neRCH dziri pedyo zvikuru, asi kwete mukati mechikamu chekuumba hukama hwakati (Mufananidzo 1, A na E). Zvisinei, panogona kunge paine hukama muC-terminal amino acids dzisina kugadziriswa dzemapuroteni maviri aya, izvo zvinogona kupa nzira yekuunganidza protein-W panguva yekuungana kweRC-LH114-W complex.
(A) Mapuroteni-W, anotarisana neLH1αβ14 muchimiro chekatuni, ane cheni yedivi yakaita setsvimbo (tsvuku), inoratidzwa muchikamu chedhiragiramu yemagetsi (ganda regrey rakajeka rine mwero we contour we 0.13). (B) Mapuroteni-W anomiririrwa nepamusoro pemavara anopikisa mvura. Nzvimbo dze polar nedzakachajwa dzinoratidzwa mu cyan, nzvimbo dzinopikisa mvura dzinoratidzwa muchena, uye nzvimbo dzinopikisa mvura zvakanyanya dzinoratidzwa muorenji. (C naD) Mapuroteni-W anomiririrwa mukatuni, kurongeka kwawo kwakafanana nekwa (A) (C), uye anotenderedzwa ne 180° (D). Zvichienderana nenzvimbo iri mu sequence, masalati anosiyanisa anoshandisa rainbow color scheme, apo N-terminal iri bhuruu uye C-terminal iri tsvuku. (E) Mapuroteni-W mumaonero akafanana nekwa (A), uye masalati ari pa interface yeprotein-W:LH1 anomiririrwa netsvimbo dzine zviratidzo zvakabatanidzwa. (F) Mapuroteni-W anotenderedzwa ne90° zvichienzaniswa ne (E) ne LH1αβ14 mumufananidzo wekatuni, uye zvichienzaniswa nezvinosara zveinterface mumufananidzo webha. Zvinosara zvakapetwa kubva ku beta polypeptide zvakanyorwa. Cofactor inoratidzwa sebhaa inoenderana neruvara rweMufananidzo 1, β-DDM yakaora inoratidzwa mugrey, uye oxygen inoratidzwa mutsvuku. (G) Maonero ari mu (F) anotenderedzwa ne180°, nezvinosara zvakakurumbira zve alpha polypeptide yakanyorwa.
Protein-W inotsiva αβ heterodimer (yechi15 muMufananidzo 1F), nokudaro ichidzivirira kuvharika kwe loop uye kutsveyamisa matatu ekutanga αβ heterodimers. Zvakaonekwa kuti kona yepamusoro yekutsveyamisa ye αβ-1 heterodimer yekutanga kana tichienzanisa nefirimu yakajairika yaive 25° kusvika 29° (Mufananidzo 1, A na E), iyo yakagadzirwa ne 2° kusvika 8° inclination ye αβ-1 muRC A sharp contrast-LH116 (Mufananidzo 1, B na F). Heterodimers yechipiri neyechitatu dzakatsveyamisa pa 12° kusvika 22° uye 5° kusvika 10°, zvichiteerana. Nekuda kwekusagadzikana kweRC, kutsveyamisa kwe αβ-1 hakusanganisire peya yechipiri ye αβ (inoenderana ne 16th αβ muMufananidzo 1F), nokudaro zvichiita mukaha wakajeka mu LH1 ring (Mufananidzo 1, A na E). Nekuda kwekushaikwa kwemaheterodimer maviri eαβ, pamwe chete nekurasikirwa kwemaBChl mana nemacarotenoid maviri, hapana kana carotenoids inosunga kune yakamonyoroka αβ-1 subunit, zvichikonzera kuti LH114-W ring ive nemacarotenoid gumi nematatu eVegetarian uye 28 BChls. Kufungidzirwa kwenzvimbo kwemacomplex maviri ari munzvimbo dzeαβ1 kusvika ku7 kwakaderera pane kwemamwe maLH1 loop, ayo anogona kuratidza plasticity yeLH1 subunit iri pedyo neRC QB site (Mufananidzo 4).
Mifananidzo yeRC-LH114-W (A naB) uye RC-LH116 (C naD) inoratidzwa kubva pamaonero akafanana ekumusoro/kurutivi (A naB) (A naC) uye pamusoro pemugodhi weMufananidzo 1. (B naD). Makiyi ane mavara anoratidzwa kurudyi.
Imwe chete core complex ine stoichiometric ratio ye1:14 iRhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Zvisinei, protein W nePufX hazvina homology yakajeka, uye zvine simba guru paLH1 structures dzavo. PufX iTMH imwe chete ine N-terminal cytoplasmic domain inosangana necytoplasmic side yeRC-H subunit (13) panzvimbo inoenderana neRps. palustris LH116αβ-16. PufX inogadzira nzira yekuchinjana kwequinone/quinolone pakati peRC-LH1 necytochrome bcl complex uye iripo mune yese Rba. sphaeroides core complex (13). Kunyangwe monomer-monomer interface iri muRba. Iyo sphaeroides RC-LH1-PufX dimer iri panzvimbo yekusunga yeprotein W muRC-LH114-W, uye mukaha unokonzerwa nePufX neprotein-W uri panzvimbo yakaenzana (Mufananidzo S7A). Mukaha uri muRC-LH114-W wakabatanawo ne quinone channel (8) yePseudomonas rosea LH1, iyo inoumbwa nema peptides asina hukama neprotein W kana PufX (Mufananidzo S7B). Pamusoro pezvo, quinone channel muBlc. Iyo emerald green LH1 inoumbwa nekusabvisa imwe γ subunit (7) iri munzvimbo yakafanana (Mufananidzo S7C). Kunyangwe ichitungamirirwa nemapuroteni akasiyana, kuoneka kweiyi quinone/quinolol channels munzvimbo yakafanana muRC-LH1 complex kunoita semuenzaniso wekushanduka-shanduka, zvichiratidza kuti mukaha unogadzirwa neprotein W unogona kushanda semugero wequinone.
Mukana uri muLH114-W loop unobvumira kuumbwa kwenzvimbo yemembrane inoenderera mberi pakati penzvimbo yemukati yeRC-LH114-W complex nemembrane yakawanda (Mufananidzo 1G), pane kubatanidza ma domain maviri kuburikidza ne protein pore sezviri muma protein. Iyo RC-LH116 complex yakafanana ne closed Tch. Needle-like complex (22) (Mufananidzo 1H). Sezvo kupararira kwe quinone kuburikidza ne membrane kuchikurumidza kupfuura kupararira kuburikidza ne protein channel yakamanikana, loop yakavhurika yeLH114-W inogona kubvumira RC turnover nekukurumidza kupfuura loop yakavharirwa yeLH116, uye kupararira kwe quinone muRC kunogona kuderedzwa zvakanyanya. Kuti tiedze kana protein W ichikanganisa kushandurwa kwe quinones kuburikidza neRC, takaita cytochrome oxidation assay pane imwe concentration ye ubiquinone 2 (UQ2) (analogue ye natural UQ10 ine shorter isoprene tail) (Mufananidzo 2E). Kunyangwe kuvapo kwequinone yakacheneswa kuchikanganisa kutsanangurwa kwakarurama kwechiratidzo cheMichaelis (RC-LH114-W naRC-LH116 zvakakodzera 0.2±0.1μM uye 0.5±0.2μM, zvichiteerana), chiyero chepamusoro cheRC-LH114-W (4.6±0.2 e-RC-1 s-1) chakakura ne28±5% kupfuura RC-LH116 (3.6±0.1 e-RC-1 s-1).
Pakutanga takafungidzira kuti protein-W inowanikwa muinenge 10% ye core complex (16); pano, huwandu hwevanhu vari mumasero ekukura kwemasero ane chiedza chidiki, chepakati nepakati, uye ane chiedza chakakwirira huri 15±0.6%, 11±1% uye 0.9±0.5, zvichiteerana % (Mufananidzo 2F). Kuenzanisa kwehuwandu hwemass spectrometry kwakaratidza kuti kuwedzerwa kwehistidine tag hakuna kuderedza huwandu hweprotein-W zvichienzaniswa nemarudzi emhando yemusango (P = 0.59), saka mazinga aya haasi chinhu cheprotein-W yakachinjwa (Mufananidzo S10). Zvisinei, kugara kweprotein-W muRC-LH1 complex uku kunogona kubvumira mamwe maRC kuti ashanduke nekukurumidza, nokudaro zvichideredza kuchinjana kwequinone/quinolone zvishoma nezvishoma muRC-LH116 complex. Takaona kuti huwandu hwevanhu vari muchiedza chakanyanya hahuenderani nedata re transcriptomics razvino, iro rinoratidza kuti pufW gene expression inowedzera pasi pechiedza chakasimba (Mufananidzo S11) (23). Musiyano uripo pakati pe pufW transcription ne protein-W incorporation mu RC-LH1 complex unovhiringidza uye unogona kuratidza kurongeka kwakaoma kweprotein.
MuRC-LH114-W, mamorekuru matanhatu ecardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) uye 29 β-DDM akagoverwa uye akaumbwa maari maCDL matanhatu, 24 POPC, 2 POPG uye 12 βDDM. RC-LH116 (Mufananidzo 5, A naB). Mumaumbirwo maviri aya, CDL inenge iri kudivi re cytoplasmic re complex, nepo POPC, POPG ne β-DDM zvakanyanya kuwanikwa kudivi re luminal. Mamorekuru maviri e lipid ne detergent akaparadzaniswa munzvimbo ye αβ-1 kusvika ku αβ-6 ye RC-LH114-W complex (Mufananidzo 5A), uye mashanu akaparadzaniswa munzvimbo yakaenzana ye RC-LH116 (Mufananidzo 5B). Malipid akawanda akawanikwa kune rimwe divi re complex, kunyanya CDL, akaunganidzwa pakati peRC neαβ-7 kusvika kuαβ-13 (Mufananidzo 5, A naB). Mamwe malipid nema detergent akagadziriswa zviri kunze kweLH1 ring, uye acyl chains dzakagadziriswa zvakanaka dzinotambanuka pakati peLH1 subunits, dzakanyorwa seβ-DDM muRC-LH114-W, uye dzinotsanangurwa seβ-DDM muRC A musanganiswa weβ-DDM nePOPC-LH116. Nzvimbo dzakafanana dze chelating lipids nema detergents muchimiro chedu dzinoratidza kuti inzvimbo dzakabatana dzakakosha mumuviri (Mufananidzo S12A). Nzvimbo dzemamorekuru akaenzana muTch dzinewo kugadzikana kwakanaka. Nyoro uye Trv. Strain 970 RC-LH1s (Mufananidzo S12, B kusvika E) (9, 12) uye hydrogen-bonding residues yeboka re lipid head zvakaratidza kuchengetedza kwakanaka mukurongeka kwezvikamu (Mufananidzo S13), zvichiratidza kuti Conserved CDL inobatanidza neRC (24), nzvimbo idzi dzinogona kuchengetedzwa muRC-LH1 complex.
(A naB) Mapeptidi eRC-LH114-W (A) naRC-LH116 (B) anomiririrwa nemakatuni, uye pigments dzinomiririrwa nemarodhi, uchishandisa ruvara rwuri muMufananidzo 1. Mafuta anoratidzwa mutsvuku, uye sipo dzinoratidzwa mugrey. UQ yakabatana nenzvimbo dzeRC QA neQB ndeyero, nepo UQ yakaparadzana iri yebhuruu. (C naD) Maonero akafanana na (A) na (B), ane mafuta akasiyiwa. (E kusvika G) Maonero akakura eQ1(E), Q2(F) naQ3(G) kubva kuRC-LH116, ane cheni dzepadivi dzinopesvedzerana. Mabond ehydrogen anoratidzwa semitsetse mitema yakapatsanurwa.
MuRC-LH116, RC QA neQB UQ, zvose zviri zviviri, zvinotora chikamu mukufambiswa kwemaerekitironi mukupatsanurwa kwechaja, zvinoora munzvimbo dzazvo dzekubatanidza. Zvisinei, muRC-LH114-W, QB quinone haisati yagadziriswa uye ichakurukurwa zvakadzama pazasi. Pamusoro peQA neQB quinones, mamorekuru maviri eUQ ane chelated (ari pakati peRC neLH1 rings) akaiswa muRC-LH114-W structure zvichienderana nemapoka avo emusoro akanyatsogadziriswa (ari muQ1 neQ2, zvichiteerana). Mufananidzo 5C). Mayuniti maviri eisoprene akapihwa kuQ1, uye density map inogadzirisa miswe gumi yakazara yeisoprene yeQ2. Muchimiro cheRC-LH116, mamorekuru matatu eUQ10 ane chelated (Q1 kusvika Q3, Mufananidzo 5D) akagadziriswa, uye mamorekuru ese ane density yakajeka mumuswe wese (Mufananidzo 5, D kusvika G). Mumaumbirwo maviri aya, nzvimbo dzemapoka emusoro wequinone eQ1 neQ2 dzine kuenderana kwakanaka (Mufananidzo S12F), uye dzinongobatana neRC chete. Q1 iri pamusuwo weW gap yeRC-LH114-W (Mufananidzo 1G ne5, C, D naE), uye Q2 iri pedyo nenzvimbo yekubatanidza QB (Mufananidzo 5, C, D) naF). Masara eL-Trp143 neL-Trp269 akachengetedzwa ari pedyo zvikuru neQ1 neQ2 uye anopa mukana wekudyidzana kweπ-stacking (Mufananidzo 5, E naF, uye Mufananidzo S12). L-Gln88, 3.0 Å kubva ku distal oxygen yeQ1, inopa hydrogen bond yakasimba (Mufananidzo 5E); masara aya akachengetedzwa mumaRC ese kunze kwehukama huri kure zvakanyanya (Mufananidzo S13). L-Ser91 inotsiviwa neThr mune mamwe maRC mazhinji (Mufananidzo S13), i3.8 Angstroms kubva kumethyl oxygen yeQ1, uye inogona kupa hydrogen bonds isina simba (Mufananidzo 5E). Q3 inoita seisina hukama chaihwo, asi iri munzvimbo inonyungudika pakati peRC-M subunit neLH1-α subunit 5 kusvika 6 (Mufananidzo 5, D naG). Q1, Q2 neQ3 kana maquinones ari pedyo akagadziriswawo muTch. Gentle, Trv. Strain 970 neBlc. Chimiro cheiris (9, 10, 12) chinonongedzera kunzvimbo yekuchengetedza quinone binding muRC-LH1 complex (Mufananidzo S12G). MaUQ mashanu akaora muRC-LH116 anowirirana zvakanaka ne5.8±0.7 ye complex yega yega inosarudzwa ne high performance liquid chromatography (HPLC), nepo maUQ matatu akaora muRC-LH114-W ari pasi pane. Kukosha kwakayerwa kwe6.2±0.3 (Mufananidzo S14) kunoratidza kuti kune maUQ molecules asina kugadziriswa muchimiro.
Mapolypeptide eL neM epseudo-symmetric imwe neimwe ine maTMH mashanu uye inoumba heterodimer inosanganisa BChl dimer imwe, maBChl monomers maviri, mabacterial (BPh) monomers maviri, uye imwe isiri Heme iron uye imwe kana maviri maUQ10 molecules. Kuburikidza nekuvapo kwehydrogen bonds pa terminal ketone group uye kuunganidzwa kwayo kunozivikanwa muRps, macarotenoids anobatanidzwa muM-subunit, iyo inonzi cis-3,4-dehydroorhodopin. Mhando (25). Nzvimbo yekunze yeRC-H yakasungirirwa kumembrane neTMH imwe chete. Chimiro cheRC chakafanana netatu subunit RC yemarudzi ane hukama (akadai seRba). sphaeroides (PDB ID: 3I4D). Macrocycles eBChl neBPh, carotenoid backbone ne non-heme iron zvinopindirana mukati me resolution range yezvivakwa izvi, sezvinoita boka reUQ10 head panzvimbo yeQA uye QB quinone paRC-LH116 (Mufananidzo S15).
Kuwanikwa kwezvivakwa zviviri zveRC zvine mwero wakasiyana wekugara panzvimbo yeQB kunopa mukana mutsva wekuongorora shanduko dzinoenderana neQB quinone binding. MuRC-LH116 complex, QB quinone iri panzvimbo yakabatana zvizere (26), asi kupatsanurwa kweRC-LH114-W hakuna QB quinone. Hapana QB quinone muRC-LH114-W, izvo zvinoshamisa nekuti complex inoshanda, kupfuura RC-LH116 complex ine QB quinone yakagadziriswa nemaumbirwo. Kunyangwe mhete mbiri dzeLH1 dzichienderana nequinones nhanhatu, shanu dzakagadziriswa nemaumbirwo muRC-LH116 ring yakavharwa, nepo nhatu chete dzakaganhurirwa nemaumbirwo muRC-LH114-W ring yakavhurika. Kusagadzikana kwechimiro uku kwakawedzera kunogona kuratidza kukurumidza kutsiviwa kweRC-LH114-W QB sites, kukurumidza kwequinone kinetics mucomplex, uye kuwedzera mukana wekuyambuka LH1 loop. Tinokurudzira kuti kushaikwa kweUQ panzvimbo yeRC QB yeRC-LH114-W kungave kuri mugumisiro wenzvimbo yakaoma uye inoshanda zvakanyanya, uye nzvimbo yeQB yeRC-LH114-W yakavharwa pakarepo mukuchinjana kweUQ Danho chairo (kupinda panzvimbo yeQB kwavharwa) rinoratidza chimiro chechiitiko ichi.
Pasina QB, kutenderera kwakabatana kweL-Phe217 kunzvimbo isingaenderane neUQ10 binding, nekuti ichakonzera kugongana kwenzvimbo nechikamu chekutanga cheisoprene chemuswe (Mufananidzo 6A). Pamusoro pezvo, shanduko huru dzechimiro dziri pachena, kunyanya helix de (pfupi helix iri mu loop pakati peTMH D naE) uko L-Phe217 inotamisirwa kupocket yeQB binding uye kutenderera kweL-Tyr223 (Mufananidzo 6A) Kuputsa chisungo chehydrogen neM-Asp45 framework uye kuvhara suo renzvimbo yeQB binding (Mufananidzo 6B). Helix de pivots pahwaro hwayo, Cα yeL-Ser209 inotamisirwa ne0.33Å, nepo L-Val221Cα ichitamisirwa ne3.52Å. Hapana shanduko dzinoonekwa muTMH D naE, dzinogona kugoneka muzvivakwa zvese (Mufananidzo 6A). Sekuziva kwedu, ichi ndicho chimiro chekutanga muRC yechisikigo chinovhara nzvimbo yeQB. Kuenzanisa nechimiro chakazara (chakasungwa neQB) kunoratidza kuti quinone isati yaderedzwa, shanduko yechimiro inodiwa kuti ipinde muquinone. L-Phe217 inotenderera kuti igadzire kudyidzana kweπ-stacking neboka remusoro wequinone, uye helix inoshanduka ichienda kunze, zvichibvumira skeleton yeL-Gly222 necheni yedivi yeL-Tyr223 kugadzira network yehydrogen bond ine chimiro chehydrogen bond chakagadzikana (Mufananidzo 6, A naC).
(A) Katuni inopindirana yehologram (L chain, orenji/M chain, magenta) uye apo (grey) structure, umo ma key residues anoratidzwa muchimiro chekumiririra kwakafanana netsvimbo. UQ10 inomiririrwa nebhara reyero. Mutsetse une madotsi unoratidza hydrogen bonds dzakaumbwa muchimiro chose. (B naC) Kumiririrwa kwepamusoro kweapolipoprotein nechimiro chose cherin'i, zvichiratidza oxygen yecheni yedivi yeL-Phe217 mubhuruu uye L-Tyr223 mutsvuku, zvichiteerana. L subunit yeorenji; M naH subunits hazvina ruvara. (D naE) Apolipoprotein (D) uye whole (E) RC QB sites [ruvara na (A) zvichiteerana] uye Thermophilus thermophilus PSII (girini, bhuruu ine quinone yepurasitiki; PDB ID: 3WU2) Align (58).
Zvisingatarisirwi, kunyange zvazvo maumbirwo akati wandei eRC dzisina QB dzisina LH1 dziripo, shanduko dzakaonekwa muchidzidzo ichi hadzina kumbotaurwa kare. Izvi zvinosanganisira chimiro chekupera kweQB kubva kuBlc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) uye Rba. sphaeroides (PDB ID: 1OGV) (29), ese akafanana nechimiro chavo cheQB. Kuongorora kwakanyatsoitwa kwe3PRC kwakaratidza kuti mamorekuru edetergent eLDAO (Lauryl Dimethyl Amine Oxide) anosunga pamusuwo wenzvimbo yeQB, izvo zvinogona kudzivirira kugadziriswazve kuita chimiro chakavharwa. Kunyangwe LDAO isingaori panzvimbo imwechete mu1EYS kana 1OGV, maRC aya anogadzirwa achishandisa sipo imwechete uye nekudaro anogona kugadzira mhedzisiro imwechete. Chimiro chekristaro cheRba. Sphaeroides RC yakabatana ne cytochrome c2 (PDB ID: 1L9B) inoita kunge ine nzvimbo yakavharwa yeQB. Zvisinei, muchiitiko ichi, nzvimbo yeN-terminal yeRC-M polypeptide (inoshanda nenzvimbo yekubatanidza yeQB kuburikidza neH bond yeTyr residue paQ helix) inotora chimiro chisina kujairika, uye shanduko yeQB conformational haisati yaongororwa zvakanyanya (30). Chinonyaradza ndechekuti hatina kuona rudzi urwu rwekuchinja kweM polypeptide muRC-LH114-W structure, iyo yakafanana neN-terminal region yeRC-LH116 RC. Zvinofanirawo kucherechedzwa kuti mushure mekubviswa kwedetergent-based LH1 antenna, apolipoprotein RCs muPDB dzakagadziriswa, izvo zvakabvisa internal quinone pools uye lipids mumukaha uri pakati peRC nepamusoro peLH1 ring yakapoteredza (31, 32). RC inoramba ichishanda nekuti inochengetedza zvese zvinoumba makemikari, kunze kweQB quinone inoora, iyo isina kugadzikana zvakanyanya uye inowanzo rasikirwa panguva yekugadzirira (33). Pamusoro pezvo, zvinozivikanwa kuti kubviswa kweLH1 uye natural cyclic lipids kubva kuRC kunogona kukanganisa mashandiro, akadai sehupenyu hupfupi hweP+QB-state yakaparadzaniswa nechaji (31, 34, 35). Saka, tinofungidzira kuti kuvapo kweLH1 ring yemuno yakakomberedza RC kunogona kuchengetedza nzvimbo "yakavharwa" yeQB, nokudaro kuchengetedza nharaunda yemuno iri pedyo neQB.
Kunyangwe apolipoprotein (isina QB quinone) uye chimiro chakazara zvichingomiririra mifananidzo miviri chete yekutendeuka kwenzvimbo yeQB, pane zviitiko zvakatevedzana, pane zviratidzo zvekuti kubatana kunogona kuvharwa kudzivirira kubatanazve nehydroquinone Kudzivirira kudziviswa kwe substrate. Kudyidzana kwequinolol nequinone pedyo nenzvimbo yeQB yeapolipoprotein kunogona kunge kwakasiyana, izvo zvinoita kuti irambwe neRC. Zvagara zvichitaurwa kuti shanduko dzechimiro dzinoita basa mukusunga nekuderedza maquinone. Kugona kwemaRC akaomeswa kuderedza maquinone mushure mekugadziriswa kwerima kwakanganiswa (36); X-ray crystallography inoratidza kuti kukuvara uku kunokonzerwa nemaquinone eQB akavharirwa muchimiro che "distal" anenge 4.5 Å kubva panzvimbo inoshanda yeproximal (26), 37). Tinokurudzira kuti chimiro ichi chekubatanidza distal chinoratidza mamiriro ari pakati peapolipoprotein nechimiro chemhete yakazara, iyo inotevera kusangana kwekutanga nequinone nekuvhurwa kwenzvimbo yeQB.
Rudzi rwechipiri rweRC rwunowanikwa muPSII complex yemamwe mabhakitiriya ephototrophic uye cyanobacteria, algae nemiti ine kuchengetedzwa kwechimiro uye mashandiro (38). Kurongeka kwechimiro kunoratidzwa muMufananidzo 6 (D naE) kunosimbisa kufanana pakati pePSII RCs nenzvimbo yeQB yebhakitiriya reRC complex. Kuenzanisa uku kwagara kuri muenzaniso wekudzidza masisitimu ane hukama hwepedyo ekubatanidza nekuderedza quinone. Mabhuku apfuura akaratidza kuti shanduko dzechimiro dzinoperekedzwa nekuderedza quinones kwePSII (39, 40). Nokudaro, tichifunga nezvekuchengetedzwa kweRC, nzira iyi yekusunga isina kuonekwa kare inogonawo kushanda kune nzvimbo yeQB yePSII RC mumiti yephototrophic ine oxygen.
Rps ΔpufW (kubviswa kwepufW kusina zita) uye PufW-His (C-terminal 10x His-tagged protein-W inoburitswa kubva kumhando dzepufW locus). palustris CGA009 yakatsanangurwa mubasa redu rekare (16). Mhando idzi ne isogenic wild-type parent dzakatorwa kubva mufiriji nekuburitsa masero mashoma paPYE (imwe neimwe 5 g litre -1) (yakachengetwa muLB pa -80 °C, ine 50% ( w/v) glycerol) protein, yeast extract uye succinate) agar [1.5% ( w/v)] plate. Plate iyi yakachengetwa husiku hwese murima patembiricha yemumba pasi pemamiriro ekusashanda zvakanaka, uye yakavhenekerwa nechiedza chichena (~50 μmolm-2 s-1) chakapihwa neOSRAM 116-W halogen bulbs (RS Components, UK) kwemazuva matatu kusvika mashanu kusvika koloni imwe chete yaonekwa. Koroni imwe chete yakashandiswa kubaya 10 ml yeM22+ medium (41) yakawedzerwa ne0.1% (w/v) casamino acids (inozonzi M22). Kukura uku kwakakura mumamiriro ekunze eoxygen shoma murima pa34°C nekuzunguzwa pa180 rpm kwemaawa makumi mana nemasere, uye ipapo 70 ml yekukura uku yakabayiwa mumamiriro ezvinhu akafanana kwemaawa makumi maviri nemana. Kukura kwesemi-aerobic kune vhoriyamu ye1 ml kunoshandiswa kubaya 30 ml yeM22 medium mubhodhoro regirazi re30 ml reuniversal screw-top transparent uye rakapiswa nemwaranzi (~50μmolm-2 s-1) kwemaawa makumi mana nemasere ne sterile magnetic force Stirring rod. Zvadaro 30 ml yekukura uku yakabayiwa ne 1 litre yekukura pasi pemamiriro akafanana, iyo yakazoshandiswa kubaya marita angangoita mapfumbamwe ekukura akavhenekerwa pa ~200 μmolm-2 s-1 kwemaawa makumi manomwe nemaviri. Masero akatorwa ne centrifugation pa 7132 RCF kwemaminitsi makumi matatu, akamiswazve mu ~10 ml ye 20 mM tris-HCl (pH 8.0), ndokuchengetwa pa -20°C kusvika zvichidikanwa.
Mushure mekunyunguduka, wedzera mamwe makristaro e deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) uye mapiritsi maviri eRoche holoenzyme protease inhibitor (Merck, UK) kumasero akadzorerwa. Mu 20,000 psi French pressure cell (Aminco, USA), masero akakanganiswa ka8 kusvika ku12. Mushure mekubvisa masero asina kupwanyika uye marara asinganyunguduke ne centrifugation pa 18,500 RCF kwemaminitsi gumi nemashanu pa 4°C, membrane yakabviswa kubva ku lysate ine pigment ne centrifugation pa 113,000 RCF kwemaawa maviri pa 43,000°C. Rasa chidimbu chinonyunguduka uye dzorera membrane ine mavara mu 100 kusvika 200 ml ye 20 mM tris-HCl (pH 8.0) uye sanganisa kusvika pasina ma aggregates anooneka. Kamudzira kakasungirirwa kakaiswa mu 20 mM tris-HCl (pH 8.0) (Anatrace, USA) kaine 2% (w/v) β-DDM kweawa imwe murima pa4°C nekukurunga zvinyoro nyoro. Wobva waisa centrifuge pa70°C kuti unyungudise 150,000 RCF pa4°C kweawa imwe chete kuti ubvise zvasara zvisinganyunguduke.
Kamudzira kanonyungudutsa kubva muchikamu cheΔpufW kakaiswa pachikamu che50 ml cheDEAE Sepharose ion exchange column ine mavhoriyamu matatu (CV) e-binding buffer [20 mM tris-HCl (pH 8.0) ine 0.03% (w / v) β-DDM]. Gezai koramu ne-CV binding buffers mbiri, mozogeza koramu ne-binding buffers mbiri dzine 50 mM NaCl. Iyo RC-LH116 complex yakabviswa ne-linear gradient ye150 kusvika 300 mM NaCl (mu-binding buffer) pa1.75 CV, uye yasara binding complex yakabviswa ne-binding buffer ine 300 mM NaCl pa0.5 CV. Unganidza spectrum yekunyudzwa kwemvura iri pakati pe250 ne1000 nm, chengeta fraction ine absorbance ratio (A880/A280) yakakura kupfuura 1 pa880 kusvika 280 nm, sanganisa kaviri mu binding buffer, uye shandisa maitiro akafanana zvakare pa DEAE column On purification. Sanganisa fractions ne A880/A280 ratios yepamusoro pe 1.7 uye A880/A805 ratios yepamusoro pe 3.0, ita third round ye ion exchange, uye chengeta fractions ne A880/A280 ratios yepamusoro pe 2.2 uye A880/A805 ratios yepamusoro pe 5.0. Chikamu chakacheneswa zvishoma chakaiswa mu ~2 ml mufirita yeAmicon 100,000 molecular weight cut-off (MWCO) centrifugal (Merck, UK), ndokuiswa paSuperdex 200 16/600 size exclusion column (GE Healthcare, US) ine 200 mM NaCl buffer, uye yobva yabviswa mubuffer imwechete pa1.5 CV. Unganidza maspectra ekunyudzwa kwechikamu chekunyudzwa kwehukuru, uye simbisa maspectra ekunyudzwa neA880/A280 ratios pamusoro pe2.4 uye A880/A805 ratios pamusoro pe5.8 kusvika 100 A880, uye pakarepo shandisai iwo pakugadzirira cryo-TEM grid kana kuchengetera. Chengetai pa -80°C kusvika panodiwa.
Kamudzira kanonyungudutsa kubva kuPufW-His kakaiswa pa20 ml HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) ine 200 mM NaCl uye 0.03% (w/w)) muIMAC buffer (GE Healthcare). v) β-DDM]. Koramu yakagezwa neCV shanu dzeIMAC buffer, uyezve neCV shanu dzeIMAC buffer dzine 10 mM histidine. Core complex yakabviswa kubva pakoramu neIMAC buffer shanu dzine 100 mM histidine. Chikamu chine RC-LH114-W complex chakaiswa kusvika ~10 ml mutangi rakasanganiswa rakagadzirwa neAmicon 100,000 MWCO filter (Merck, UK), yakasanganiswa ka20 nebinding buffer, uye yozowedzerwa ku25 ml. Mukoramu yeDEAE Sepharose, maCV mana akasungirirwa kubuffer anoshandiswa pachine nguva. Gezai koramu nemabuffer mana eCV binding, mobva mabvisa complex pamaCV masere pamutsetse we0 kusvika 100 mM NaCl (mubinding buffer), uye mamwe maCV mana asara ane 100 mM binding buffer. Macomplex akasara akabviswa pasodium chloride pamwe chete neA880/A280 ratio yakakwira kupfuura 2.4 uye A880/A805 ratio yakakwira kupfuura 4.6 fractions akaiswa kusvika ~2 ml muAmicon 100,000 MWCO centrifugal filter, uye akazadzwa ne1.5 CV IMAC pamberi Buffer yakaenzana Superdex 200 16/600 size exclusion column, uye obva abviswa mubuffer imwechete pamusoro pe1.5 CV. Unganidza maspectra ekunyudzwa kwezvikamu zvisingabatanidzwi nehukuru uye simbisa maspectra ekunyudzwa neA880/A280 ratios pamusoro pe2.1 uye A880/A805 ratios pamusoro pe4.6 kusvika 100 A880, ayo anoshandiswa ipapo ipapo pakugadzirira TEM grid yakaomeswa kana kuchengetwa pa -80°C kusvika painodiwa.
Muchina weLeica EM GP wekuisa mufiriji wakashandiswa kugadzira magridi eTEM ane tembiricha yakaderera. Muchina uyu wakasanganiswa neIMAC buffer kusvika paA880 ye50, uye 5μl yakazoiswa pamesh yemhangura yeQUANTIFOIL 1.2/1.3 yakaputirwa necarbon (Agar Scientific, UK). Isai grid pa20°C uye 60% hunyoro hwemasekonzi makumi matatu, mozoidzima kwemaminitsi matatu, mozoidzima ne ethane yemvura pa -176°C.
Data reRC-LH114-W complex rakarekodhwa pa eBIC (Electronic Bioimaging Center) (British Diamond Light Source) neTitan Krios microscope, iyo inoshanda nevoltage inokurumidza ye300kV, ine nominal magnification ye130,000× uye simba re- Sarudza gap ye20 eV. Gatan 968 GIF Quantum ine K2 peak detector yakashandiswa kurekodha mifananidzo mu counting mode kuunganidza data. Saizi yepixel yakaganhurirwa i1.048Å, uye chiyero chedosi i3.83 e-Å-2s-1. Ndakaunganidza bhaisikopo mumasekonzi gumi nerimwe ndikaripatsanura kuita zvikamu makumi mana. Shandisa nzvimbo yakaputirwa nekabhoni kuti utarisezve microscope, uye wozounganidza mafirimu matatu paburi rega rega. Pakazara, mafirimu 3130 akaunganidzwa, aine defocus values ​​​​pakati pe -1 ne -3μm.
Data reRC-LH116 complex rakaunganidzwa pachishandiswa maikorosikopu imwecheteyo paAsterbury Biostructure Laboratory (University of Leeds, UK). Data rakaunganidzwa nenzira yekuverenga nekuwedzera kwe130 k, uye saizi yepikiseli yakaganhurirwa kusvika 1.065 Å nedosi ye4.6 e-Å-2s-1. Bhaisikopo rakarekodhwa mumasekonzi gumi nemaviri uye rakakamurwa kuita zvikamu makumi mana nemasere. Pakazara, mafirimu 3359 akaunganidzwa, aine defocus values ​​​​pakati pe -1 ne -3μm.
Kugadziriswa kwese kwedata kunoitwa muRelion 3.0 pipeline (42). Shandisa Motioncorr 2 (43) kugadzirisa kufamba kwebeam nekuyerwa kwedosi, wobva washandisa CTTFIND 4.1 (44) kuti uone parameter yeCTF (contrast transfer function). Maphotomicrographs akajairika mushure mematanho ekutanga ekugadzirisa aya anoratidzwa muMufananidzo 2. S16. Template yekusarudza otomatiki inogadzirwa nekusarudza nemaoko mapikiseti angangoita 250 e1000 particles mufuremu ye250-pixel uye isina reference two-dimensional (2D) classification, nokudaro ichiramba maclassifications iwayo anosangana nekusvibiswa kwesampuli kana asina hunhu hunoonekwa. Zvadaro, sarudzo otomatiki yakaitwa pama microphotographs ese, uye RC-LH114-W yaive 849,359 particles, uye RC-LH116 complex yaive 476,547 particles. Zvidimbu zvese zvakasarudzwa zvakapinda muzvikamu zviviri zve2D zvisina kutaurwa, uye mushure mekumhanya kwega kwega, zvidimbu zvinosangana nenzvimbo yekabhoni, kusvibiswa kwemuenzaniso, kusina zvinhu zvakajeka kana zvidimbu zvinopindirana zvakanyanya zvinorambwa, zvichikonzera kuti 772,033 (90.9%) uye 359,678 (75.5%) ) Zvidimbu zvinoshandiswa muzvikamu zve3D zveRC-LH114-W neRC-LH116 zvakateerana. Muenzaniso wekutanga we3D reference wakagadzirwa uchishandisa nzira yestochastic gradient descent. Uchishandisa muenzaniso wekutanga sereferensi, zvidimbu zvakasarudzwa zvakakamurwa muzvikamu zvina mu3D. Uchishandisa muenzaniso uri muchikamu ichi sereferensi, ita 3D refining pazvidimbu zviri muchikamu chikuru, wobva washandisa 15Å low-pass filter yekutanga kufukidza nzvimbo inonyungudutsa, wedzera 6 pixels yemipendero yakapfava, uye mushure mekugadzirisa mapixel kugadzirisa basa reGatan K2 peak Modulation transfer re detector yepamusoro. Kune data reRC-LH114-W, modhi yekutanga iyi yakagadziriswa nekubvisa density yakasimba pamucheto we mask (yakabviswa kubva ku core complex density muUCSF Chimera). Mamodheru akabuda (resolutions dzeRC-LH114-W neRC-LH116 dziri 3.91 ne4.16 Å, zvichiteerana) anoshandiswa sereferensi ye round yechipiri ye 3D classification. Ma particles anoshandiswa akaiswa mu class yekutanga ye 3D uye haana hukama hwakasimba nenzvimbo. Anopindirana kana kushaya ma structure features ari pachena. Mushure me round yechipiri ye 3D classification, category ine resolution yepamusoro yakasarudzwa [Kune RC-LH114-W, category imwe i 377,703 particles (44.5%), ye RC-LH116, kune ma categories maviri, anosvika 260,752 particles (54.7%) , Kwaanenge akafanana chete kana akaenzana mushure mekutenderera kwekutanga nemusiyano mudiki]. Zvidimbu zvakasarudzwa zvinotorwazve mubhokisi remapikiseli mazana mana uye zvinonatswa ne3D refining. Solvent mask inogadzirwa uchishandisa 15Å low-pass filter yekutanga, 3 pixel map expansion uye 3 pixel soft mask. Uchishandisa per-particle CTF refining, per-particle motion correction uye second round ye per-particle CTF refining, 3D refining, solvent masking uye post-processing zvinoitwa mushure medanho rega rega kuti zvinyatso gadzirisa texture inobuda. Uchishandisa FSC (Fourier Shell Correlation Coefficient) cut-off value ye0.143, resolutions yemamodheru ekupedzisira eRC-LH114-W neRC-LH116 i2.65 ne2.80Å, zvichiteerana. FSC curve yemodheru yekupedzisira inoratidzwa muMufananidzo 2. S17.
Mapuroteni ese akatevedzana anotorwa kubva kuUniProtKB: 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); Mapuroteni-W (PufW; UniProt ID: Q6N1K3). SWISS-MODEL (45) yakashandiswa kugadzira modhi yeRC, ine mapuroteni akatevedzana eRC-L, RC-M uye RC-H uye chimiro chekristaro cheRba. sphaeroides RC yakashandiswa se template (PDB ID: 5LSE) (46). Shandisa chishandiso che "fit map" muUCSF Chimera kuti ukwane muenzaniso wakagadzirwa nemepu (47), vandudza chimiro cheprotein, uye cofactor [4×BChl a (zita re residue residue residue remonomer library = BCL), 2×BPh a (BPH), rudzi rumwe kana maviri eUQ10 (U10), imwe non-heme iron (Fe) uye imwe 3,4-dihydrohexacarbonylcholine (QAK)] shandisa Coot (48) kuwedzera. Sezvo QAK isipo mu monomer library, yakagadziriswa uchishandisa eLBOW tool muPHENIX (49).
Zvadaro, LH1 subunit yakavakwa. Pakutanga, chishandiso chekuvaka otomatiki muPHENIX (49) chakashandiswa kugadzira otomatiki chikamu cheLH1 sequence uchishandisa mepu uye LH1-α uye LH1-β protein sequences se input. Sarudza LH1 subunit yakakwana, ibvise woiisa muCoot, wedzera nemaoko sequence isipo mairi, uye gadzirisa nemaoko chimiro chose usati wawedzera maBCls maviri (BCL) uye spirilloxanthin (CRT) [zvichienderana neRps yakakodzera The density yeLH1 complex uye carotenoid content inozivikanwa. Species (17)]. Kopa LH1 subunit yakazara, uye shandisa UCSF Chimera "Docking Map Tool" kuti uise padock munzvimbo iri pedyo isiri yemuenzaniso yeLH1 density, wozoigadzirisa muCoot; dzokorora maitiro acho kusvika LH1 subunits dzese dzagadzirwa. Kune chimiro cheRC-LH114-W, nekubvisa density isina kurongeka muCoot, protein inokamurwa kubva kune zvimwe zvikamu zvisiri zveprotein zviri muUSCF Chimera map uye chishandiso cheAutobuild chinoshandiswa kugadzira modhi yekutanga, uye zvimwe zvikamu zvidiki (protein-W) Modeling. MuPHENIX (49). Wedzera chero zvikamu zvisipo kumodhi inobuda muCoot (48), wobva wagadzirisa subunit yese nemaoko. Density isina kurongeka inokodzera musanganiswa wemafuta (PDB monomer library ID yeCDL = CDL, POPC = 6PL uye POPG = PGT), β-DDM detergent (LMT) uye mamorekuru eUQ10 (U10). Shandisa PHENIX optimization (49) uye manual optimization muCoot (48) kuti ugadzirise modhi yekutanga yese kusvika nhamba dzemodhi uye kunaka kwekuona kwe fit zvisisakwanise kuvandudzwa. Chekupedzisira, shandisa LocScale (50) kurodza mepu yemuno, wobva waita mamwe ma cycles ekuenzanisa density isina kurongeka uye automatic uye manual optimization.
Mapeptides, macofactors nemamwe mafuta nemaquinones akaiswa mukati mehuwandu hwawo anoratidzwa muMifananidzo 1 ne2. S18 kusvika S23. Ruzivo rwehuwandu hwemuenzaniso wekupedzisira rwunoratidzwa muTafura S1.
Kunze kwekunge zvataurwa neimwe nzira, maUV/Vis/NIR absorption spectra akaunganidzwa paCary60 spectrophotometer (Agilent, USA) pa1 nm intervals kubva pa250 nm kusvika 1000 nm uye nguva yekubatanidza ye0.1s.
Sanganisai sampuro mu quartz cuvette ine nzira ye 2 mm kuenda ku A880 ye 1, mounganidza spectrum yekunyudzwa iri pakati pe 400 ne 1000 nm. Ma circular dichroic spectra akaunganidzwa paJasco 810 spectropolarimeter (Jasco, Japan) pa 1 nm intervals iri pakati pe 400 nm ne 950 nm pa scan rate ye 20 nm min-1.
Chiyero chekupera kwemvura (molar extinction coefficient) chinoonekwa nekudurura core complex kuita A880 yeinenge 50. Dururira 10μl volume mu 990μl binding buffer kana methanol, wobva waunganidza absorption spectrum nekukasika kuti uderedze BChl degradation. Zviri muBChl yemuenzaniso wega wega we methanol zvakaverengerwa ne extinction coefficient pa 771 nm ye 54.8 mM-1 cm-1, uye extinction coefficient yakaongororwa (51). Govanisa huwandu hweBChl hwakayerwa ne 32 (RC-LH114-W) kana 36 (RC-LH116) kuti uone huwandu hwe core complex, iyo inoshandiswa kuona absorption spectrum yemuenzaniso mumwe chete wakaunganidzwa mu buffer Extinction coefficient. parallel. Zviyero zvitatu zvakadzokororwa zvakatorwa pamuenzaniso wega wega, uye avhareji yekunyudzwa kweBChl Qy maximum yakashandiswa pakuverenga. Chiyero chekutsakatika kweRC-LH114-W chakayerwa pa878 nm i3280±140 mM-1 cm-1, nepo chiyero chekutsakatika kweRC-LH116 chakayerwa pa880 nm chiri 3800±30 mM-1 cm-1.
UQ10 yakayerwa zvichienderana nenzira iri mu (52). Muchidimbu, reverse phase HPLC (RP-HPLC) yakaitwa uchishandisa Agilent 1200 HPLC system. Nyungudutsa inenge 0.02 nmol yeRC-LH116 kana RC-LH114-W mu50μl ye50:50 methanol:chloroform ine 0.02% (w/v) ferric chloride, uye isa jekiseni reBeckman Coulter Ultrasphere ODS 4.6 mm Nyungudutsa mu 1 ml-1 min-1 pa40°C muHPLC solvent (80:20 methanol:2-propanol) pa ×25 cm column. Ita isocratic elution muHPLC solvent kuti utarise absorbance pa 275 nm (UQ10), 450 nm (carotenoids) uye 780 nm (BChl) kweawa imwe chete. Chiyero chikuru mu 275 nm chromatogram pamaminitsi makumi maviri nemashanu nemashanu chakasanganiswa, icho chakanga chisina mamwe macompounds anowanikwa. Nzvimbo yakabatanidzwa inoshandiswa kuverenga huwandu hwemolar hweUQ10 hwakabviswa maererano ne calibration curve yakaverengerwa kubva pakuisirwa kwe pure standards kubva pa0 kusvika pa5.8 nmol (Mufananidzo S14). Sampuro yega yega yakaongororwa mumakopi matatu, uye kukanganisa kwakataurwa kunoenderana neSD yeavhareji.
Mhinduro ine RC-LH1 complex ine Qy absorption yepamusoro ye0.1 yakagadzirwa ne 30 μM reduced horse heart cytochrome c2 (Merck, UK) uye 0 kusvika 50 μMUQ2 (Merck, UK). Samples nhatu dze 1-ml dzakagadzirwa pane imwe neimwe UQ2 concentration uye dzakaiswa husiku hwose murima pa 4°C kuitira kuti dzigadziriswe zvizere murima risati rayerwa. Mhinduro yacho yakaiswa muOLIS RSM1000 modular spectrophotometer ine 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm middle uye 0.6 mm outlet slits. Firita ye600 nm long pass inoiswa pamusuwo we phototube yemuenzaniso uye reference photomultiplier tube kuti isaburitse chiedza chekufadza. Absorbance yakatariswa pa 550 nm nenguva yekubatanidza ye 0.15 s. Mwenje wekufadza unoburitswa kubva ku880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) kuburikidza netambo yefiber optic ine 90% intensity kuburikidza neDC2200 controller (Thorlabs Ltd., UK) uye unoburitswa kune chiedza pakona ye90° of. Danda rekuyera rinopesana negirazi kuti ridzose chero chiedza chisina kutorwa nemuenzaniso pakutanga. Tarisa kunyudzwa kwechiedza 10 s usati wavheneka kwe50 s. Zvadaro kunyudzwa kwechiedza kwakatariswazve kwe60 s murima kuti uongorore kuti quinolol inoderedza sei cytochrome c23 + (ona Mufananidzo S8 kuti uwane data raw).
Data rakagadziriswa nekuisa chiyero chekutanga chemutsetse mukati memasekondi 0.5 kusvika ku10 (zvichienderana nehuwandu hweUQ2) uye kuyera huwandu hwesampuro dzese nhatu pahuwandu hwega hwega hweUQ2. Hunhu hweRC-LH1 hwakaverengerwa nechiyero chekupera kwemvura hwakashandiswa kushandura chiyero kuita catalytic efficiency, hwakarongwa muOrigin Pro 2019 (OriginLab, USA), uye hwakaiswa kuMichaelis-Menten model kuti ione hunhu hweKm neKcat.
Pakuyerwa kwemushonga we transient absorption, sampuli yeRC-LH1 yakasanganiswa kusvika ~2μM muIMAC buffer ine 50 mM sodium ascorbate (Merck, USA) uye 0.4 mM Terbutin (Merck, USA). Ascorbic acid inoshandiswa semupi wema electron anobayira, uye tert-butaclofen inoshandiswa seQB inhibitor kuona kuti mupi wema RC anoramba akaderera (ndiko kuti, kwete photooxidized) mukati mekuita kwekuyera. Inenge 3 ml yesampuli inowedzerwa kune custom rotating cell (inenge 0.1 m mu dhayamita, 350 RPM) ine 2 mm optical path length kuti ive nechokwadi chekuti sampuli iri munzira ye laser ine nguva yakakwana yekugadzirisa rima pakati pe excitation pulses. Shandisa ~100-fs laser pulses kuti uwedzere Ti: Sapphire laser system (Spectra Physics, USA) kuti usimudzire sampuli pa880 nm pa repetition rate ye1 kHz (20 nJ yeNIR kana 100 nJ yeVis). Usati waunganidza data, isa sample muchiedza chekuyedza kwemaminitsi angangoita makumi matatu. Kuonekwa kwacho kuchaita kuti QA isashande (zvichida kuderedza QA kamwe chete kana kaviri). Asi ndapota cherechedza kuti maitiro aya anodzoreka nekuti mushure menguva yakareba yekuchinja kwerima, RC inodzokera zvishoma nezvishoma kuQA activity. Helios spectrometer (Ultrafast Systems, USA) yakashandiswa kuyera transient spectra nenguva yekunonoka ye -10 kusvika 7000 ps​​. Shandisa Surface Xplorer software (Ultrafast Systems, USA) kupatsanura data sets, wozobatanidza woita standardize. Shandisa CarpetView software package (Light Conversion Ltd., Lithuania) kushandisa combined data set kuti uwane differential spectra ine chekuita nekuora, kana kushandisa function inosanganisira multiple exponents ne instrument response kuti ikwane single-wavelength spectral evolution muOrigin (OriginLab, USA).
Sezvambotaurwa pamusoro apa (53), firimu rephotosynthetic rine LH1 complex risina RC ne peripheral LH2 antenna rakagadzirwa. Kamudzira kakasanganiswa mu 20 mM tris (pH 8.0) ndokuzoiswa mu quartz cuvette ine nzira ye 2 mm optical. 30nJ laser pulse yakashandiswa kukurudzira sampuli pa 540 nm nenguva yekunonoka ye -10 kusvika 7000 ps. Gadzira data set sezvakatsanangurwa kune Rps. pal sample.
Kamudzira kakaiswa mu centrifugation pa 150,000 RCF kwemaawa maviri pa 4°C, uye ipapo kunyudzwa kwaro pa 880 nm kwakadzoserwa mu 20 mM tris-HCl (pH 8.0) uye 200 mM NaCl. Nyungudutsa kamudzira nekukurunga zvishoma nezvishoma mu 2% (w/v) β-DDM kweawa imwe murima pa 4°C. Sampuro yakasanganiswa mu 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) kusvika pahuwandu hweprotein hwe 2.5 mg ml-1 (Bio-Rad analysis). Kugadziriswa kwakawedzerwa kwakaitwa kubva panzira yakamboburitswa (54), kutanga nekuderedzwa kwe 50 μg protein kuita huwandu hwe 50 μl TEAB ine 1% (w/v) sodium laurate (Merck, UK). Mushure mekuita sonication kwemasekonzi makumi matanhatu, yakaderedzwa ne5 mM tris(2-carboxyethyl)phosphine (Merck, UK) pa37°C kwemaminitsi makumi matatu. PaS-alkylation, isa sample ne10 mM methyl S-methylthiomethanesulfonate (Merck, UK) woiisa kubva mu200 mM isopropanol stock solution kwemaminitsi gumi patembiricha yemumba. Kugayiwa kweproteolytic kwakaitwa nekuwedzera 2 μg trypsin/endoproteinase Lys-C mixture (Promega UK) ndokuiswa pa37°C kwemaawa matatu. Laurate surfactant yakabviswa nekuwedzera 50 μl ethyl acetate uye 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) uye vortexing kwemasekonzi makumi matanhatu. Kupatsanurwa kwechikamu kwakasimudzirwa ne centrifugation pa 15,700 RCF kwemaminitsi mashanu. Zvichienderana neprotocol yemugadziri, C18 spin column (Thermo Fisher Scientific, UK) yakashandiswa kuspira nekunyatsobvisa munyu muchikamu chezasi chine peptide. Mushure mekuomeswa ne vacuum centrifugation, sampuro yakanyungudutswa mu 0.5% TFA uye 3% acetonitrile, uye 500 ng yakaongororwa ne nanoflow RP chromatography pamwe chete ne mass spectrometry uchishandisa system parameters dzakatsanangurwa kare.
Shandisa MaxQuant v.1.5.3.30 (56) kuti uzive mapuroteni uye uverenge huwandu hwemapuroteni kuti utsvage Rps. palustris proteome database (www.uniprot.org/proteomes/UP000001426). Data remass spectrometry proteomics rakaiswa muProteomeXchange Alliance kuburikidza nePRIDE partner repository (http://proteomecentral.proteomexchange.org) pasi pedataset identifier PXD020402.
Kuti iongororwe neRPLC pamwe chete neelectrospray ionization mass spectrometry, RC-LH1 complex yakagadzirwa kubva ku wild-type Rps. Uchishandisa nzira yakamboburitswa (16), huwandu hweprotein hwakagadzirwa mumasero epalustris hwaive 2 mg ml-1 mu 20 mM Hepes (pH 7.8), 100 mM NaCl uye 0.03% (w/v) β- (Bio-Rad analysis) ) DDM. Zvinoenderana neprotocol yemugadziri, shandisa 2D purification kit (GE Healthcare, USA) kuti ubvise 10 μg protein nenzira yeprecipitation, uye unyungudise precipitate mu 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile uye 20% (v/v) mvura. Microliters mashanu akaongororwa neRPLC (Dionex RSLC) pamwe chete nemass spectrometry (Maxis UHR-TOF, Bruker). Shandisa MabPac 1.2×100 mm column (Thermo Fisher Scientific, UK) kuti uparadzanise pa60°C uye 100μlmin -1, ine gradient ye85% (v / v) solvent A [0.1% (v / v) FA uye 0.02% (V/v) TFA aqueous solution] kusvika ku85% (v/v) solvent B [0.1% (v/v) FA uye 0.02% (v/v) mu90% (v/v) acetonitrile TFA] Uchishandisa standard electrospray ionization source uye default parameters kweanopfuura maminitsi makumi matanhatu, mass spectrometer inowana 100 kusvika 2750 m/z (mass-to-charge ratio). Nerubatsiro rweExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), gadzira mass spectrum kune ma subunits e complex.
Masero akakurira kwemaawa makumi manomwe nemaviri pasi pe100 ml NF-low (10μMm-2 s-1), yepakati (30μMm-2 s-1) kana yakakwira (300μMm-2 s-1). M22 yepakati (M22 yepakati umo ammonium sulfate inosiyiwa uye sodium succinate inotsiviwa nesodium acetate) mubhodhoro re100 ml screw-top (23). Mumasekonzi mashanu e30-s, 0.1 micron glass beads dzakarukwa ne vhoriyamu ye 1:1 kuti dzinyungudike masero uye dzitonhore pachando kwemaminitsi mashanu. Chinhu chisinganyunguduke, masero asina kupwanyika uye magirazi beads zvakabviswa ne centrifugation pa 16,000 RCF kwemaminitsi gumi mu benchtop microcentrifuge. Kamudzira kakaparadzaniswa mu rotor yeTi 70.1 ine 100,000 RCF mu 20 mM tris-HCl (pH 8.0) ine 40/15% (w/w) sucrose gradient kwemaawa gumi.
Sezvakatsanangurwa mubasa redu rekare, kuona immune tag yeHis paPufW (16). Muchidimbu, purified core complex (11.8 nM) kana membrane ine concentration yakafanana yeRC (inosarudzwa ne oxidation ichibvisa reduction difference spectrum uye ichienzanisa mutoro uri pa stained gel) mu 2x SDS loading buffer (Merck, UK) yakaderedzwa kaviri. Mapuroteni akaparadzaniswa pa replica 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). Gel yakapendwa neCoomassie Brilliant Blue (Bio-Rad, UK) kuti ikwanise kuisa uye kuona RC-L subunit. Protein iri pa gel yechipiri yakaendeswa ku methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) kuti iwane immunoassay. Kamudzira kePVDF kakavharirwa mu50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 uye 5% (w / v) mukaka wakabviswa mafuta, ndokuzoiswa mu anti-His primary antibody (mu Dilute the antibody buffer [50 mM tris-HCl (pH 7.6), 150 mM NaCl uye 0.05% (v/v) Tween-20] mu1:1000 A190-114A, Bethyl Laboratories, USA) kwemaawa mana. Mushure mekugezwa katatu kwemaminitsi mashanu mu antibody buffer, membrane yakabatanidzwa ne horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (yakasanganiswa 1:10,000 mu antibody buffer). Incubate kuti ionekwe (maminetsi mashanu mushure mekugezwa katatu mu antibody buffer) uchishandisa WESTAR ETA C 2.0 chemiluminescence substrate (Cyanagen, Italy) uye Amersham Imager 600 (GE Healthcare, UK).
Nekudhirowa kupararira kwejeri yega yega yakacheneswa kana nzira yekudzivirira muviri, kubatanidza nzvimbo iri pasi pegomo uye kuverenga chiyero chekudzivirira muviri cheRC-L (jeri yakacheneswa) neProtein-W (immunoassay), muImageJ (57) Gadzira mufananidzo. Aya mareshiyoni akachinjwa kuita molar ratios nekufungidzira kuti chiyero cheRC-L kune protein-W muRC-LH114-W sample yakachena chaive 1:1 uye kugadzirisa data rese zvinoenderana.
Kuti uwane zvimwe zvinyorwa zvechinyorwa chino, ndapota ona http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
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David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe, Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Maumbirwo emhando yepamusoro e light trap 1 complex iri pakati pe reaction centre anopa ruzivo rutsva pamusoro pe quinone dynamics.
David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe, Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Maumbirwo emhando yepamusoro e light trap 1 complex iri pakati pe reaction centre anopa ruzivo rutsva pamusoro pe quinone dynamics.
©2021 American Association for the Advancement of Science. kodzero dzose dzakachengetedzwa. AAAS ishamwari yeHINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef uye COUNTER. ScienceAdvans ISSN 2375-2548.