Mprempren†Mprempren address: OX11 0DE, UK, Diamond Building, Harwell Nyansahu ne Nneɛma Foforo Atrae, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Ɛlektrɔnik Abɔde a Nkwa Wom Mfoniniyɛbea.
Reaction center light-harvesting complex 1 (RC-LH1) ne ade titiriw a ɛma hann yɛ adwuma wɔ mmoawa a wɔyɛ hann a ɛyɛ kɔkɔɔ no mu. Yɛde cryo-electron microscopy nhyehyɛe abien a ɛwɔ RC-LH1 complex a efi Rhodopseudomonas palustris mu bae. 2.65-Å resolution nhyehyeɛ a ɛwɔ RC-LH114-W complex no mu no yɛ subunit LH1 loops 14 a atwa RC ho ahyia, a protein W na ɛtwa mu, berɛ a complex a enni protein-W no yɛ RC composition a RC atwa ho ahyia koraa. Wɔatoto 16 subunit LH1 loop mu. Saa nhyehyɛe ahorow yi a wɔde toto ho no ma yenya nhumu wɔ quinone ahoɔden a ɛwɔ RC-LH1 complex no mu, a nea ɛka ho ne conformational nsakrae a kan no na wontumi nhu bere a ɛkyekyere quinone wɔ RC QB beae no, ne beae a quinone a ɛkyekyere mmeae aboafo wɔ, a ɛboa ma wɔde wɔn kɔ RC. W protein no nhyehyɛe soronko no mma LH1 loop no nto mu, na ɛnam so ma wonya ɔkwan bi a wɔfa so ma quinone/quinolone nsakrae no yɛ ntɛmntɛm.
Ɛkame ayɛ sɛ ahoɔden a photosynthesis de ma no tumi ma nkwa a ɛwɔ asase so nyinaa kɔ so tra hɔ, na ɛwɔ tumi kɛse ma owia mu abɔde a nkwa wom ho mfiridwuma. Bere a wɔhyɛ wiase nyinaa hann a wɔde yɛ adwuma ho nkuran no, mmoawa a wɔyɛ hann a ɛyɛ kɔkɔɔ nso da ahoɔden akwan horow ne nipadua mu nneɛma ahorow adi. Wobetumi akwati photosynthesis na wɔanyin sɛ heterotrophic bacteria wɔ sum mu, wobetumi asiesie nitrogen ne carbon dioxide, ayɛ hydrogen, na wɔasɛe nneɛma a ɛyɛ huam (1-3). Sɛnea ɛbɛyɛ na wɔanya ahoɔden ama saa nneɛma yi no, ɛsɛ sɛ wɔdan hann ntɛmntɛm na wɔyɛ no yiye ma ɛbɛyɛ nnuru ahoɔden. Saa adeyɛ yi fi ase bere a hann-trapping antenna complex no twetwe hann na ɛde ahoɔden a wɔakyere no kɔ reaction center (RC), na ɛnam so fi ase charge separation (4 – 7). Nneɛma titiriw a ɛma hann yɛ adwuma wɔ mmoawa a wɔyɛ hann kɔkɔɔ mu no yɛ type 2 RC, a hann-twa complex 1 (LH1) atwa ho ahyia, na ɛyɛ RC-LH1 core complex. LH1 nam αβ heterodimers a ɛyɛ kurukuruwa a wɔahyehyɛ, a emu biara kyekyere mmoawa chlorophyll (BChl) a molecule abien ne carotenoid biako anaa abien (8-12). LH1 antena a ɛyɛ mmerɛw sen biara no yɛ αβ heterodimers 16 anaa 17 a atwa RC (9-13) ho ahyia wɔ loop a wɔato mu, nanso wɔ core complexes afoforo mu no, transmembrane peptides twa LH1 a atwa ho ahyia no kɔ so no mu , Enti ɛma quinol/quinone trɛw a ɛda RC ne cytochrome bc1 complex ntam no nya nkɔso (11, 13-15 na ɛwɔ hɔ). Afifide a ɛyɛ kɔkɔɔ a ɛma hann Rhodopseudomonas (Rps.) yɛ abɔde a ɛyɛ nhwɛso a ebetumi ate ahoɔden ne ɛlɛtrɔnik a ɛkɔ baabi foforo a ɛboa hann a ɛma hann yɛ adwuma no ase. Ahwehwɛ nhyehyɛe a edi kan a Rps. Palustris RC-LH1 complex no nhwɛso ne RC, a heterodimeric LH1 loops 15 atwa ho ahyia, a protein bi a wonnim a wɔfrɛ no “Protein W” (14) na ɛtwa mu. Akyiri yi wɔkyerɛe sɛ protein-W yɛ RPA4402, a ɛyɛ protein a ɛyɛ 10.5kDa a wonnim ne su a ɛwɔ transmembrane helices (TMH) abiɛsa a wɔahyɛ ho nkɔm ( 16 ). Yɛhyɛ nyansa sɛ wɔbɛsesa rpa4402 awosu mu abɔde a ɛkyerɛw protein W no din ayɛ no pufW na ama ɛne din a wɔde frɛ awosu mu nkwaadɔm a ɛkyerɛw RC-L, M (pufL, pufM) ne LH1α, β (pufA, pufB) subunits no ahyia. Nea ɛyɛ anigye no, protein-W wɔ RC-LH1 bɛyɛ 10% pɛ mu, na ɛda Rps adi. palustris ma RC-LH1 a ɛyɛ den ahorow abien ba. Ɛha yi, yɛbɔ amanneɛ sɛ high-resolution cryo-EM (cryo-EM) nhyehyɛe ahorow abien core complexes, biako a protein W ne 14 αβ heterodimers, biako nso nni protein W ne closed 16 Heterodimer LH1 loop. Yɛn nhyehyeɛ no gyina hɔ ma anammɔn nsakraeɛ wɔ nteaseɛ a ɛfa RC-LH1 complex a ɛwɔ Rps ho. palustris, efisɛ yɛayɛ nnipa dodow a ɛyɛ pɛ a ɛwɔ variant biara mu no mu nhwehwɛmu na yɛwɔ resolution a ɛdɔɔso a yɛde bɛma peptide biara ne pigments a wɔakyekyere ne lipids ne quinones a ɛfa ho no pefee. Saa nhyehyɛe ahorow yi a wɔde toto ho no kyerɛ sɛ TMH protein-W abiɛsa a wonhuu bi wɔ RC-LH1 complex foforo biara mu de besi nnɛ no ma quinone kwan bi ba ma quinone/quinolone nsakrae no yɛ ntɛmntɛm. Wɔahu mmeae dodow bi a wɔakora so a ɛkyekyere lipid ne quinone, na yɛada nsakrae foforo a ɛba wɔ conformational mu adi wɔ quinone ne RC a wɔaka abom akyi, a ebia ɛfata ma photosystem II (PSII) RC a oxygenated phototrophic organisms. Nea yɛahu no ma yenya nhumu foforo wɔ quinone/quinolone a ɛkyekyere ne nsakrae a ɛkɔ so wɔ RC-LH1 core complex a ɛwɔ purple phototrophic bacteria no mu no ho.
Sɛnea ɛbɛyɛ a ɛbɛyɛ mmerɛw sɛ wɔbɛyɛ nhwehwɛmu a ɛkɔ akyiri wɔ nneɛma abien a ɛyɛ den a wohu wɔ Rps. palustris, yɛde biochemical akwan yiyi RC-LH1 biara fi mu. Wɔtew protein W a enni mu (a wɔbɛfrɛ no ΔpufW wɔ ha) no ho fii ɔkwan a enni pufW awosu mu abɔde no mu ( 16 ), na RC-LH1 a ɛyɛ den biako pɛ na wobetumi ayɛ. Protein W a ɛwɔ mu no, ɔkwan bi so na ɛyɛ. Wɔde 10x His tag a ɛwɔ ne C-awiei no sesa protein W a ɛwɔ saa ɔkwan yi so no, sɛnea ɛbɛyɛ a wobetumi de protein W a ɛwɔ mu no afra protein W dodow no ara a enni mu no mu yiye denam dade a wɔbɛma ayɛ nea entumi nkɔ baabiara no so. Wɔatetew nea ɛyɛ den no mu yiye (16) Affinity Chromatography (IMAC).
Sɛnea wɔakyerɛ wɔ Mfonini 1 mu no, complex abien no nyinaa kura sub-unit RC abiɛsa (RC-L, RC-M ne RC-H) a LH1 antenna atwa ho ahyia. 2.80-A nhyehyɛe a ɛwɔ complex a protein-W nni mu no kyerɛ αβ heterodimers 16, a ɛyɛ LH1 loop a wɔato mu a atwa RC ho ahyia koraa, a wɔbɛfrɛ no wɔ ha sɛ RC-LH116 complex. 2.65Å nhyehyeɛ a ɛwɔ protein-W a ɛwɔ mu no wɔ 14-heterodimer LH1 a protein-W twa mu, a wɔbɛfrɛ no wɔ ha sɛ RC-LH114-W.
(A ne B) Nneɛma a wɔde afrafra no ho mfonini a ɛwɔ soro. (C ne D) Nneɛma a wɔde yɛ aduru a wɔakyekyere a wɔda no adi wɔ abaa mu. (E ne F) Nneɛma a ɛyɛ den a wohu fi cytoplasmic ani no wɔ peptides ne LH1 subunits a wɔde gyina hɔ ma wɔ cartoons mu, na wɔde nɔma ahyɛ dɔn no kwan so fi protein-W gap no mu [a ɛne Rba nɔma hyia. sphaeroides a ɛyɛ den (13)]. Wɔ LH1-α fam no, protein subunit no kɔla yɛ kɔkɔɔ; wɔ LH1-β fam no, protein subunit no kɔla yɛ bruu; wɔ protein-W fam no, protein no yɛ kɔkɔɔ; wɔ RC-H fam no, ɛyɛ cyan; wɔ RC-L fam no, ɛyɛ Orange; ma RC-M, a ɛyɛ magenta. Wɔde abaa gyina hɔ ma cofactors, ahabammono gyina hɔ ma BChl ne BPh a molecules, purple gyina hɔ ma carotenoids, na kɔkɔɔ gyina hɔ ma UQ10 molecules. (G ne H) Protein-W nsonsonoe a ɛwɔ RC-LH114-W complex (G) ne RC-LH116 complex (H) no fã a ɛyɛ pɛ no ho mfonini a wɔayɛ no kɛse. Wɔda cofactors adi wɔ ɔkwan a wɔfa so hyɛ ahunmu ma, wɔde chelated quinone kyerɛ wɔ bruu mu. Wɔde nsensanee bruu a ɛyɛ nsensanee a ɛwɔ (G) mu na esi protein-W nsonsonoe no so dua, na wɔde nsensanee tuntum a ɛyɛ nsensanee a ɛwɔ (H) mu na esi ntokuru nketewa a quinone/quinolol trɛw wɔ LH116 ring no so no so dua.
Mfonini 1 (A ne B) kyerɛ RC a LH1αβ heterodimers a wɔabue anaa wɔato mu atwa ho ahyia, a emu biara kyekyere BChl abien ne carotenoid biako (Mfonini 1, C ne D). Nhwehwɛmu ahorow a atwam ada no adi sɛ Rps ne LH1 complex no. Wɔ biosynthetic kwan a spirulina xanthin fa so no, saa mmoa ahorow yi kura carotenoids dodow a adi afra ( 17 ). Nanso, spiropyrroxanthin ne carotenoid titiriw na ne duru yɛ nea abotɔyam wom. Enti, yɛpaw sɛ yɛbɛyɛ spiroxanthin nhwɛso wɔ mmeae a LH1 kyekyere nyinaa. Alpha ne beta polypeptides no yɛ TMH biako a ɛwɔ akyi mmeae a ɛwɔ ntini ntiantiaa (Mfonini 1, A, B, E, ne F). Ɛwom sɛ wɔanhu sɛnea nkae 17 a ɛwɔ C-awiei no dɔɔso de, nanso wɔpaapaee alpha polypeptide no mu fii Met1 mu kɔɔ Ala46 mu wɔ nneɛma abien no nyinaa mu. Wɔtew β polypeptide so fii Gly4 kɔɔ Tyr52 wɔ RC-LH116 mu, ne Ser5 kɔɔ Tyr52 wɔ RC-LH114-W mu. Wɔanhu density biara a ɛyɛ N-terminal 3 anaa 4 anaa 13 C-terminal residues (Mfonini S1). Mass spectrometry nhwehwɛmu a wɔyɛe wɔ RC-LH1 complex a wɔadi afra a wɔasiesie afi wuram-su no mu no kyerɛe sɛ ɔmantam a ayera no fi heterologous cleavage a ɛwɔ saa peptides yi mu (Mfonini S1 ne S2). Wɔsan nso huu α-Met1 no N-terminal formylation (f). Nhwehwɛmu no kyerɛe sɛ α-peptide no yɛ nkae fMet1 kosi Asp42/Ala46/Ala47/Ala50, na β-peptide no yɛ nkae Ser2 kosi Ala53, a ɛne EM density map a ɛwɔ ɔhyew a ɛba fam no hyia yiye.
α-His29 ne β-His36 a ɛyɛ biako no ma BChls anim ne anim; αβ heterodimer biara ne n’afipamfo hyiam yɛ loop a wɔabue (RC-LH114-W) anaasɛ loop a wɔatoto mu (RC-LH116) twa RC no ho hyia The exciton coupled pigment array (Mfonini 1, C ne D). Sɛ yɛde toto 877 nm band a ɛwɔ RC-LH114-W ho a, 880 nm absorption red shift a ɛwɔ RC-LH116 mu no yɛ 3 nm (Mfonini 2A). Nanso, ɛkame ayɛ sɛ circular dichroism spectrum no yɛ pɛ (Mfonini 2B), a ɛkyerɛ sɛ ɛwom sɛ nsonsonoe a ɛda adi pefee wɔ loop a wɔabue ne nea wɔato mu ntam de, nanso BChls mpɔtam hɔ tebea no di nsɛ yiye. Ebia absorption redshift no fi ɔhyew a ɛso tew ne ahoɔden a ɛkɔ soro wɔ loop a wɔato mu no so (18, 19), nsakrae a ɛba pigment coupling mu a loop a wɔato mu no de ba (20, 21), anaasɛ saa nsunsuanso abien yi a wɔaka abom (11).
(A) Ultraviolet/visible/near-infrared absorption spectrum, a wɔde wɔn pigments a ɛne no hyia ahyɛ ne sorokɔ agyirae na wɔayɛ no sɛnea ɛte wɔ BPh peak wɔ 775 nm. (B) Kurukuruwa dichroism spectrum normalized to BChl absorbance wɔ 805 nm. (C ne D) Wɔapaw ΔA spectra afi bere-a wɔasiesie absorption spectra a ɛwɔ RC-LH114-W complex (C) ne RC-LH116 complex (D) mu. Sɛnea ɛbɛyɛ a wobetumi de atoto ho yiye no, wɔayɛ spectra nyinaa normalized to ∆A of −A wɔ 0.2 ps. (E) Cytochrome c2 oxidation dodow wɔ irradiation akyi wɔ UQ2 dodow ahorow a ɛwɔ hɔ no mu (hwɛ Mfonini S8 ma raw data). (F) Wɔ nkwammoaa a enyinii wɔ hann a ɛba fam, mfinimfini anaa kɛse ase (10, 30 anaa 300μMm-2 s-1, sɛnea ɛte biara), protein W ne RC-L subunits a ɛwɔ complex a wɔatew ho no mu ne membrane ratio a wɔatew mu no. Kyerɛ protein dodow denam SDS-polyacrylamide gel electrophoresis ne immunoassay so (hwɛ Mfonini S9 ma raw data). Kyerɛ ratio a ɛfa RC-LH114-W complex a wɔatew ho no ho. Stoichiometric ratio a RC-L ne protein-W a ɛwɔ complex no mu no yɛ 1:1.
BChls a ɛwɔ gyinabea 1 wɔ deformed αβ14 loop a ɛwɔ RC-LH114-W (Mfonini 1, A, C, ne E) no bɛn RC primary donor (P) no 6.8Å sen BChls a ɛne no sɛ wɔ RC-LH116 (Mfonini. 1, B, D, ne F, ne Mfonini S3) nanso, bere tiaa mu absorption kinetics a ɛwɔ complexes abien no mu no kyerɛ sɛ wɔ RC-LH114-W ne RC-LH116 fam no, excitation energy transfer bere constants fi LH1 kɔ RC yɛ 40 ±4 ne 44±3 ps (Mfonini 2). , C ne D, Mfonini S4 ne Table S2). Nsonsonoe titiriw biara nso nni ɛlɛtrɔnik kwan a wɔfa so de nneɛma kɔ baabi foforo wɔ RC mu (Mfonini S5 ne nsɛm a wɔde ka ho a ɛfa ho). Yɛsusu sɛ ahoɔden a wɔde fa LH1 ne RC-P ntam no hyia a ɛbɛn no firi kwan a ɛte saa ara, anim ne ahoɔden a ɛbɛtumi aba a ɛwɔ BChl dodoɔ no ara mu wɔ LH1 nhama mmienu no mu. Ɛte sɛ nea LH1 ahoɔden nhyehyɛe a wɔhwehwɛ mu de du kwan a ɛba fam koraa no nyɛ ntɛmntɛm nsen ahoɔden a wɔde fi mmeae a ɛnyɛ papa kɔ RC no tẽẽ. Open-loop LH1 loop a ɛwɔ RC-LH114-W mu no nso betumi afa ɔhyew mu kankyee a ɛho nhia mu wɔ ɔhyew a ɛba fam tebea horow mu ama nhyehyɛe mu nhwehwɛmu, na αβ14 ring conformation tenten wɔ hɔ wɔ dan mu hyew mu fi βBChls pigmentation kwan a ɛwɔ RC 1 gyinabea no.
RC-LH116 complex no kura BChls 32 ne carotenoids 16, na ne nhyehyɛe nyinaa ne nea wonya fi Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 strain ( PDB ID 7C9R) (12) ne green mu no yɛ pɛ atɛkyɛ (Blc.viridis) (PDB ID 6ET5) (10). Wɔ alignment akyi no, wohuu deviations nketenkete nkutoo wɔ αβ heterodimers gyinabea ahorow mu, titiriw 1-5, 15, ne 16 (Mfonini S6). Protein-W a ɛwɔ hɔ no nya LH1 nhyehyɛe so nkɛntɛnso kɛse. Ne TMH abiɛsa no nam nhama ntiantiaa so na ɛka bom, a N-terminal no wɔ lumen fã a ɛwɔ complex no mu na C-terminal no wɔ cytoplasmic fã (Mfonini 1A ne 3, A kosi D). Protein-W yɛ nea nsu ntumi nkɔ mu kɛse (Mfonini 3B), na TMH2 ne TMH3 ne LH1αβ-14 di nkitaho ma ɛyɛ transmembrane soro (Mfonini 3, B ne E kosi G). Nkitahodi no yɛ Phe, Leu ne Val nkae a ɛwɔ transmembrane mpɔtam hɔ titiriw. Wɔde amino acid ahorow a ɛnyɛ nsu ne αβ-14 aduru a wɔde yɛ nneɛma a ɛyɛ fɛ na ɛboaboa nneɛma a aka yi ano. Polar nkaeɛ bi nso boa ma nkitahodi no ba, a hydrogen nkitahodi a ɛda W-Thr68 ne β-Trp42 ntam wɔ abura a ɛyɛ den no ani ka ho (Mfonini 3, F ne G). Wɔ cytoplasm no ani no, Gln34 bɛn keto kuw a ɛyɛ αβ-14 carotenoids no. Bio nso, wɔsiesiee n-dodecyl β-d-maltoside (β-DDM) molecule no, na ne dua a ɛtwetwe nsu no trɛw kɔɔ protein-W ne αβ-14 ntam, na ebia lipid dua no wɔ nipadua no mu. Yɛsan nso hyɛɛ no ​​nsow sɛ C-terminal resolution regions a ɛwɔ protein W ne RCH mu no bɛn yiye, nanso ɛnyɛ nea ɛwɔ nkitahodi pɔtee a ɛbɛhyehyɛ mu (Mfonini 1, A ne E). Nanso, ebia nkitahodi ahorow bi wɔ protein abien yi C-awie amino acid ahorow a wonnya nsiesiee no mu, a ebetumi ama wɔanya ɔkwan a wɔbɛfa so agye protein-W bere a wɔreboaboa RC-LH114-W a ɛyɛ den no ano.
(A) Protein-W, a ɛhwɛ nkitahodi a ɛne LH1αβ14 wɔ cartoon kwan so no, wɔ nkɔnsɔnkɔnsɔn a ɛte sɛ abaa (kɔkɔɔ), a wɔada no adi wɔ electrostatic potential diagram no fã bi (transparent grey surface a contour level yɛ 0.13). (B) Protein-W a wɔde kɔla a ɛnyɛ nsu gyina hɔ ma. Wɔde cyan akyerɛ mmeae a polar ne charged wom, wɔde fitaa ada mmeae a nsu ntumi nkɔ mu, na wɔde borɔdɔma ada mmeae a nsu ntumi nkɔ mu denneennen adi. (C ne D) Protein-W a wɔde ayɛ mfonini wɔ cartoon mu, ne kwankyerɛ yɛ pɛ sɛnea ɛwɔ (A) (C), na ɛkyinkyini 180° (D). Sɛnea gyinabea a ɛwɔ ntoatoaso no mu kyerɛ no, nkae a wotumi hu no fa nyankontɔn kɔla nhyehyɛe, baabi a N-awiei yɛ bruu na C-awiei yɛ kɔkɔɔ. (E) Protein-W wɔ adwene koro no ara mu sɛnea ɛwɔ (A), ne nkae a ɛwɔ protein-W:LH1 ntam no, wɔde abaa a agyiraehyɛde ahorow abata ho na egyina hɔ ma. (F) Wɔdannan protein-W 90° sɛ wɔde toto (E) ne LH1αβ14 ho wɔ cartoon gyinabea no mu, na wɔde toto interface residues a ɛwɔ bar gyinabea no ho. Wɔakyerɛw nkae a ɛsensɛn beta polypeptide no mu no so. Wɔde cofactor no akyerɛ sɛ bar a ɛne Mfonini 1 kɔla hyia, wɔde akutu akyerɛ β-DDM a aporɔw no, na wɔde kɔkɔɔ akyerɛ oxygen no. (G) Wɔadannan adwene a ɛwɔ (F) no mu 180°, a alpha polypeptide a wɔakyerɛw so no nkae a ɛda nsow ka ho.
Protein-W si αβ heterodimer ananmu (nea ɛto so 15 wɔ Mfonini 1F mu), na ɛnam so siw loop a wɔbɛto mu no ano na ɛdan αβ heterodimers abiɛsa a edi kan no. Wɔhwɛɛ sɛ αβ-1 heterodimer a edi kan no anim a ɛkɔ soro sen biara bere a wɔde toto film normal no ho no yɛ 25° kosi 29° (Mfonini 1, A ne E), a ɛnam αβ-1 a ɛkɔ fam 2° kosi 8° wɔ RC A sharp contrast-LH116 mu (Mfonini 1, B ne F) so na ɛbae. Heterodimers a ɛtɔ so mmienu ne mmiɛnsa no akɔ fam wɔ 12° kɔsi 22° ne 5° kɔsi 10°, sɛdeɛ ɛteɛ biara. Esiane steric akwanside a ɛwɔ RC mu nti, αβ-1 a ɛdannan no nka αβ abien a ɛto so abien (a ɛne αβ a ɛto so 16 a ɛwɔ Mfonini 1F mu no hyia) ho, ma enti ɛma nsonsonoe a ɛda adi pefee wɔ LH1 ring no mu (Mfonini 1, A ne E) . Esiane sɛ αβ heterodimers abien nni hɔ, a BChl anan ne carotenoids abien a wɔhwere ka ho nti, carotenoids no mu biara nkyekyere αβ-1 subunit a wɔakyinkyim no, na ɛde LH114-W ring a carotenoids 13 Vegetarian ne BChls 28 wom ba. Local resolution akontabuo a ɛfa complexes mmienu a ɛwɔ αβ1 kɔsi 7 mpɔtam hɔ no sua sene deɛ ɛwɔ LH1 loop nkaeɛ no mu, a ɛbɛtumi ada plasticity a ɛwɔ LH1 subunit a ɛbɛn RC QB site no mu no adi (Mfonini 4)
Wɔde RC-LH114-W (A ne B) ne RC-LH116 (C ne D) mfonini ahorow no akyerɛ fi soro hwɛ/afã hwɛbea koro no ara (A ne B) (A ne C) ne cavity surface a ɛwɔ Mfonini 1. (B ne D). Wɔakyerɛ nsafe a ɛwɔ kɔla ahorow no wɔ nifa so.
Su foforo biako pɛ a ɛyɛ core complex a stoichiometric ratio yɛ 1:14 ne Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Nanso, protein W ne PufX nni nsɛso a ɛda adi pefee, na ɛwɔ nkɛntɛnso kɛse wɔ wɔn LH1 nhyehyɛe ahorow so. PufX yɛ TMH biako a ɛwɔ N-terminal cytoplasmic domain a ɛne cytoplasmic fã a ɛwɔ RC-H subunit (13) no di nkitaho wɔ beae a ɛne Rps hyia. palustris LH116αβ-16 na ɛwɔ hɔ. PufX yɛ ɔkwan a wɔfa so sesa quinone/quinolone wɔ RC-LH1 ne cytochrome bcl complex ntam na ɛwɔ Rba nyinaa mu. sphaeroides core a ɛyɛ den (13). Ɛwom sɛ monomer-monomer ntamgyinafo no wɔ Rba mu de. Sphaeroides RC-LH1-PufX dimer no wɔ beae a protein W kyekyere wɔ RC-LH114-W mu, na nsonsonoe a PufX ne protein-W de ba no wɔ beae a ɛyɛ pɛ (Mfonini S7A). Ɔkwan a ɛda RC-LH114-W mu no nso ne Pseudomonas rosea LH1 a wɔde peptide ahorow a ɛne protein W anaa PufX nni abusuabɔ na ɛyɛ no quinone kwan (8) a wɔde susuw ho no hyia (Mfonini S7B). Bio nso, quinone kwan a ɛwɔ Blc. LH1 a ɛyɛ ahabammono a ɛyɛ emerald a wɔde γ subunit biako (7) a wɔayi afi mu no wɔ beae a ɛte saa ara (Mfonini S7C). Ɛwom sɛ protein ahorow na ɛyɛ ntamgyinafo de, nanso ɛte sɛ nea quinone/quinolol akwan yi a ɛda adi wɔ gyinabea koro wɔ RC-LH1 a ɛyɛ den no mu no yɛ adannandi a ɛkɔ so hyia ho nhwɛso, na ɛkyerɛ sɛ ebia nsonsonoe a protein W de ba no bɛyɛ adwuma sɛ quinone kwan.
Ɔkwan a ɛda LH114-W loop no mu no ma kwan ma wɔyɛ membrane region a ɛkɔ so wɔ RC-LH114-W complex no mu ahunmu ne bulk membrane no ntam (Mfonini 1G), sen sɛ wɔbɛka domain abien no abom denam protein pore so sɛnea ɛte wɔ protein ahorow mu no. RC-LH116 complex no te sɛ Tch a wɔato mu. Nneɛma a ɛte sɛ nnade a ɛyɛ den (22) (Mfonini 1H). Esiane sɛ quinone a ɛtrɛw fa membrane no mu no yɛ ntɛmntɛm sen nea ɛtrɛw fa protein kwan teateaa no mu nti, LH114-W loop a wɔabue no betumi ama RC dannan ntɛmntɛm sen LH116 loop a wɔato mu no, na ebia quinone a ɛtrɛw kɔ RC no mu no bɛyɛ nea anohyeto wom kɛse. Sɛnea ɛbɛyɛ a yɛbɛsɔ ahwɛ sɛ ebia protein W nya quinones nsakraeɛ so nkɛntɛnsoɔ denam RC so no, yɛyɛɛ cytochrome oxidation nhwehwɛmu wɔ ubiquinone 2 (UQ2) (abɔdeɛ mu UQ10 analogue a ɛwɔ isoprene dua tiawa) dodoɔ bi so (Mfonini 2E). Ɛwom sɛ chelated quinone a ɛwɔ hɔ no siw Michaelis daa a ɛda adi sɛ ɛbɛkyerɛ pɛpɛɛpɛ (RC-LH114-W ne RC-LH116 fata ma 0.2±0.1μM ne 0.5±0.2μM, sɛnea ɛte biara), nanso RC-LH114-W dodow a ɛsen biara ( 4.6±0.2 e-RC-1 s-1) yɛ 28±5% a ɛsõ sen RC-LH116 (3.6±0.1 e-RC-1 s-1).
Yɛdii kan buu akontaa sɛ protein-W wɔ bɛyɛ 10% wɔ core complex no mu ( 16 ); ha no, nkwammoaa a ɛnyin a hann sua, hann a ɛwɔ mfimfini, ne nea hann kɛse wom no dodow yɛ 15±0.6%, 11±1% ne 0.9±0.5, sɛnea ɛte biara (Mfonini 2F). Ntotoe dodow a wɔde totoo mass spectrometry ho no kyerɛe sɛ histidine tag a wɔde kaa ho no antew protein-W dodow a ɛwɔ hɔ no so bere a wɔde totoo wuram-su ahorow ho no (P = 0.59), enti saa dodow yi nyɛ protein-W a wɔasesa no ayɛ (Mfonini S10). Nanso, protein-W a ɛba fam yi a ɛwɔ RC-LH1 complex no mu yi betumi ama RC ahorow bi adan ntɛmntɛm, na ɛnam so ama quinone/quinolone a wɔsesa brɛoo wɔ RC-LH116 complex no mu no so atew. Yɛhyɛɛ no ​​nsow sɛ hann dodow a ɛkɔ soro no ne nnansa yi transcriptomics data no nhyia, a ɛkyerɛ sɛ pufW gene expression kɔ soro wɔ hann a ano yɛ den ase (Mfonini S11) (23). Nsonsonoe a ɛda pufW nkyerɛwee ne protein-W a wɔde ka RC-LH1 a ɛyɛ den no ho no yɛ nea ɛyɛ basaa na ebetumi ada protein no nhyehyɛe a ɛyɛ den no adi.
Wɔ RC-LH114-W mu no, wɔkyekyɛ cardiolipin (CDL) 6, phosphatidylcholine (POPC) 7), phosphatidylglycerol (POPG) 1 ne β-DDM molecule 29 na wɔyɛ ho nhwɛso wɔ mu 6 CDLs, 24 POPCs, 2 POPGs ne 12 βDDMs. RC-LH116 (Mfonini 5, A ne B). Wɔ saa nhyehyɛe abien yi mu no, ɛkame ayɛ sɛ CDL wɔ cytoplasmic fã a ɛwɔ complex no mu, bere a POPC, POPG ne β-DDM dodow no ara wɔ luminal fã no. Wɔyii lipid ne detergent molecule mmienu wɔ αβ-1 kɔsi αβ-6 mpɔtam a ɛwɔ RC-LH114-W complex no mu (Mfonini 5A), na wɔyii anum wɔ RC-LH116 mpɔtam a ɛne no yɛ pɛ no mu (Mfonini 5B ). Wohuu srade pii wɔ complex no fã foforo, titiriw CDL, a ɛboaboa ano wɔ RC ne αβ-7 ntam kosi αβ-13 (Mfonini 5, A ne B). Lipids ne detergents afoforo a wɔasiesie no yiye wɔ LH1 ring no akyi, na acyl nkɔnsɔnkɔnsɔn a wɔasiesie no yiye trɛw wɔ LH1 subunits ntam, a wɔakyerɛ ase sɛ β-DDM wɔ RC-LH114-W mu, na wɔakyerɛ ase sɛ β-DDM wɔ RC A β-DDM ne POPC-LH116 afrafra mu. Gyinabea a ɛte saa ara a chelating lipids ne detergents wɔ wɔ yɛn nhyehyɛe mu no kyerɛ sɛ ɛyɛ mmeae a ɛkyekyere a ɛfa nipadua ho (Mfonini S12A). Gyinabea a molecule ahorow a ɛyɛ pɛ wɔ Tch mu nso wɔ nhyiam pa. Ɔbrɛ ne Trv. Strain 970 RC-LH1s (Mfonini S12, B kosi E) (9, 12) ne hydrogen-bonding residues a ɛwɔ lipid ti kuw no mu no kyerɛɛ sɛ wɔakora so yiye koraa wɔ sequence alignment no mu (Mfonini S13), a ɛkyerɛ sɛ Conserved CDL a ɛkyekyere RC (24), saa mmeae yi betumi akora so wɔ RC-LH1 complex no mu.
. UQ a ɛkyekyere RC QA ne QB mmeae no yɛ kɔkɔɔ, bere a UQ a ​​atew ne ho no yɛ bruu. (C ne D) Adwene koro no ara a ɛwɔ (A) ne (B), a wɔayi srade afi mu. (E kosi G) Q1(E), Q2(F) ne Q3(G) a wɔatrɛw mu afi RC-LH116, a nkɔnsɔnkɔnsɔn a ɛwɔ nkyɛnkyɛn a ɛwɔ nkɛntɛnso wɔ wɔn ho wɔn ho so. Wɔakyerɛ hydrogen nkitahodi ahorow no sɛ nsensanee tuntum a wɔabɔ no nsensanee.
Wɔ RC-LH116 mu no, RC QA ne QB UQ nyinaa a wɔde wɔn ho hyɛ ɛlɛtrɔnik a wɔde kɔ baabi foforo mu wɔ charge mpaapaemu nhyehyɛe no mu no porɔw wɔ wɔn mmeae a ɛkyekyere no. Nanso, wɔ RC-LH114-W mu no, wɔansiesie QB quinone na wɔbɛka ho asɛm kɔ akyiri wɔ ase ha. QA ne QB quinones akyi no, wɔakyekyɛ UQ molecule abien a wɔayɛ no chelated (a ɛwɔ RC ne LH1 nkaa ntam) wɔ RC-LH114-W nhyehyɛe no mu sɛnea wɔn ti akuw a wɔasiesie no yiye (a ɛwɔ Q1 ne Q2 mu, sɛnea ɛte biara) te. kwan). Mfonini 5C). Wɔde isoprene unit abien ama Q1, na density map no siesie isoprene dua 10 a edi mũ a ɛwɔ Q2 no. Wɔ RC-LH116 nhyehyɛe mu no, wɔsiesiee UQ10 molecule abiɛsa a wɔayɛ no chelated (Q1 kosi Q3, Mfonini 5D), na molecule no nyinaa wɔ density a ɛda adi pefee wɔ dua no nyinaa mu (Mfonini 5, D kosi G). Wɔ nhyehyeɛ mmienu no mu no, Q1 ne Q2 quinone ti akuo no gyinabea wɔ nhyiamu a ɛkyɛn so (Mfonini S12F), na wɔne RC nko ara na ɛdi nkitaho. Q1 wɔ RC-LH114-W W gap no ano (Mfonini 1G ne 5, C, D ne E), na Q2 bɛn beae a QB kyekyere (Mfonini 5, C, D) ne F). L-Trp143 ne L-Trp269 nkaeɛ a wɔakora so no bɛn Q1 ne Q2 yie na ɛma π-stacking nkitahodiɛ a ɛbɛtumi aba (Mfonini 5, E ne F, ne Mfonini S12). L-Gln88, 3.0 Å fi oxygen a ɛwɔ akyirikyiri a ɛwɔ Q1 no, ma hydrogen nkitahodi a emu yɛ den (Mfonini 5E); saa nkaeɛ yi na wɔkora so wɔ RC nyinaa mu gye abusuabɔ a ɛwɔ akyirikyiri paa (Mfonini S13). L-Ser91 yɛ conservatively substituted for Thr wɔ RC afoforo dodow no ara mu (Mfonini S13), ɛyɛ 3.8 Angstroms fi methyl oxygen a ɛwɔ Q1, na ebetumi ama hydrogen nkitahodi a ɛyɛ mmerɛw (Mfonini 5E). Ɛte sɛ nea Q3 nni nkitahodi pɔtee bi, na mmom ɛwɔ beae a nsu nni mu a ɛda RC-M subunit ne LH1-α subunit 5 kosi 6 ntam (Mfonini 5, D ne G). Wɔasiesie Q1, Q2 ne Q3 anaa chelated quinones a ɛbɛn hɔ nso wɔ Tch. Ɔdwoɔ, Trv. Nsu 970 ne Blc. Iris nhyehyeɛ (9, 10, 12) no twe adwene si quinone boafoɔ a ɛkyekyere beaeɛ a wɔakora so wɔ RC-LH1 complex no mu (Mfonini S12G). UQ anum a aporɔw wɔ RC-LH116 mu no ne 5.8±0.7 a ɛwɔ complex biara a wɔde high performance liquid chromatography (HPLC) kyerɛ no hyia yiye, bere a UQ abiɛsa a aporɔw wɔ RC-LH114-W mu no sua sen The measured value of 6.2±0.3 (Fig. S14) kyerɛ sɛ UQ molecules a wonnya nsiesiee wɔ nhyehyɛe no mu.
Pseudo-symmetric L ne M polypeptides no mu biara kura TMH anum na ɛyɛ heterodimer a ɛka BChl dimer biako, BChl monomers abien, bacteriophage (BPh) monomers abien, ne dade biako a ɛnyɛ Heme ne UQ10 molecule biako anaa abien bom. Ɛdenam hydrogen nkitahodi a ɛwɔ ketone kuw a etwa to no so ne nea wonim sɛ ɛboaboa ano wɔ Rps mu so no, wɔde carotenoids ka M-subunit no ho, a wɔtoo din cis-3,4-dehydroorhodopin. Nnomaa ahorow (25). RC-H akyi membrane domain no nam TMH biako so na ɛkyekyere membrane no. RC nhyehyɛe no nyinaa te sɛ subunit RC abiɛsa a ɛwɔ mmoa ahorow a ɛne wɔn ho wɔn ho di nkitaho (te sɛ Rba) no. sphaeroides (PDB ID: 3I4D) a ɛma mogya tu. BChl ne BPh, carotenoid akyi dompe ne dadeɛ a ɛnyɛ heme no macrocycles no ka bom wɔ saa nhyehyɛɛ yi resolution range mu, sɛdeɛ UQ10 ti kuw a ɛwɔ QA beaeɛ ne QB quinone a ɛwɔ RC-LH116 (Mfonini S15) no yɛ no.
RC nhyehyeɛ mmienu a ɛwɔ hɔ a ɛsono QB beaeɛ a wɔte mu no ma hokwan foforɔ a wɔde bɛhwehwɛ nsakraeɛ a ɛkɔ so daa wɔ conformational a ɛka QB quinone binding ho no mu. Wɔ RC-LH116 complex no mu no, QB quinone wɔ “proximal” gyinabea a wɔakyekyere no koraa ( 26 ), nanso RC-LH114-W a wɔpaapae mu no nni QB quinone. QB quinone biara nni RC-LH114-W mu, a ɛyɛ nwonwa efisɛ complex no yɛ adwuma, sen RC-LH116 complex a ɛwɔ structurally resolved QB quinone. Ɛwom sɛ LH1 nkaa abien no chelate bɛyɛ quinones asia de, nanso anum yɛ nhyehyɛe a wɔasiesie wɔ RC-LH116 nkaa a wɔato mu no mu, bere a abiɛsa pɛ na ɛwɔ nhyehyɛe anohyeto wɔ RC-LH114-W nkaa a wɔabue mu no mu . Saa nhyehyɛe mu ɔhaw a ɛkɔ soro yi betumi ada RC-LH114-W QB mmeae a wɔsesa ntɛmntɛm, quinone kinetics ntɛmntɛm wɔ complex no mu, ne sɛnea ɛbɛyɛ yiye sɛ wobetwa LH1 loop no akɔ soro no adi. Yɛhyɛ nyansa sɛ UQ a ​​enni RC QB beae a ɛwɔ RC-LH114-W no betumi ayɛ nea efi complex a ɛyɛ den na ɛyɛ nnam kɛse mu aba, na QB beae a RC-LH114-W wɔ no ayɛ nwini ntɛm ara wɔ UQ turnover mu The specific stage (wɔato QB site no kwan a ɛkɔ mu no mu) kyerɛ sɛnea saa dwumadi yi te.
Sɛ QB nni hɔ a, L-Phe217 a ɛka ho no a ɛkyinkyini kɔ gyinabea a ɛne UQ10 a ɛkyekyere no nhyia, efisɛ ɛbɛma ne dua no isoprene unit a edi kan no abɔ wɔ ahunmu (Mfonini 6A). Afei nso, nsakrae titiriw a ɛda adi pefee wɔ conformational mu no da adi, titiriw helix de (helix tiawa a ɛwɔ loop a ɛda TMH D ne E ntam) a wɔdan L-Phe217 kɔ QB binding kotoku no mu ne L-Tyr223 a ɛkyinkyini (Mfonini 6A ) Sɛnea ɛbɛyɛ a wobebubu hydrogen bond no ne M-Asp45 framework no na wɔato entrance a ɛwɔ QB binding site no mu (Mfonini 6B) no. Helix de pivots wɔ ne nnyinaso, Cα a ɛwɔ L-Ser209 no sesa 0.33Å, bere a L-Val221Cα no sesa 3.52Å. Nsakrae biara nni TMH D ne E a wotumi hu, a wotumi de gu so wɔ nhyehyɛe abien no nyinaa mu (Mfonini 6A). Sɛnea yenim no, eyi ne nhyehyɛe a edi kan wɔ abɔde mu RC mu a ɛto QB beae no. Sɛ wɔde toto nhyehyɛe a edi mũ (QB-bound) ho a, ɛkyerɛ sɛ ansa na wɔbɛtew quinone no so no, ɛho hia sɛ wɔyɛ nsakrae wɔ conformational mu na ama akɔ quinone no mu. L-Phe217 di akɔneaba ma ɛyɛ π-stacking nkitahodi ne quinone ti kuw no, na helix no dan kɔ abɔnten, na ɛma L-Gly222 nnompe ne L-Tyr223 afã nkɔnsɔnkɔnsɔn no yɛ hydrogen bond network a ɛwɔ hydrogen bond nhyehyɛe a ɛyɛ den (Mfonini 6, A ne C).
(A) Hologram (L nkɔnsɔnkɔnsɔn, borɔdɔma/M nkɔnsɔnkɔnsɔn, magenta) ne apo (akuturuku) nhyehyɛe a ɛka bom, a wɔda nkae atitiriw no adi wɔ ɔkwan a ɛte sɛ poma so. Wɔde dua kɔkɔɔ gyina hɔ ma UQ10. Ntrɛwmu a ɛyɛ nsensanee no kyerɛ hydrogen nkitahodi ahorow a wɔayɛ wɔ ɔdan no nyinaa mu. (B ne C) Apolipoprotein ne ring nhyehyɛe no nyinaa ho mfonini a ɛwɔ soro, a ɛtwe adwene si ɔfã nkɔnsɔnkɔnsɔn oxygen a ɛwɔ L-Phe217 mu wɔ bruu ne L-Tyr223 wɔ kɔkɔɔ mu, sɛnea ɛte biara. L subunit no yɛ borɔdɔma; M ne H subunits no nni kɔla. (D ne E) Apolipoprotein (D) ne mũ (E) RC QB mmeae [kɔla denam (A) so] ne Thermophilus thermophilus PSII (ahabammono, bruu a wɔde plastic quinone ka ho; PDB ID: 3WU2) Fa hyia (58).
Nea wɔnhwɛ kwan no, ɛwom sɛ RC ahorow a QB nnim a enni LH1 no nhyehyɛe ahorow pii wɔ hɔ de, nanso wɔmmɔ nsakrae a ɛbaa conformational a wohui wɔ nhwehwɛmu yi mu no ho amanneɛ kan. Eyinom bi ne QB depletion nhyehyɛe a efi Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) ne Rba. sphaeroides (PDB ID: 1OGV) (29), a ɛkame ayɛ sɛ ne nyinaa ne wɔn QB nhyehyɛe nyinaa yɛ pɛ. 3PRC mu nhwehwɛmu a wɔyɛe yiye daa no adi sɛ LDAO (Lauryl Dimethyl Amine Oxide) detergent molecules kyekyere wɔ QB gyinabea no ano, na ebetumi asiw nhyehyɛe foforo a wɔbɛsan ayɛ no conformation a wɔato mu no ano. Ɛwom sɛ LDAO nsɛe wɔ beae koro wɔ 1EYS anaa 1OGV mu de, nanso wɔde aduru koro no ara na ɛyɛ saa RC ahorow yi ma enti ebetumi ama nkɛntɛnso koro no ara aba. Ahwehwɛ nhyehyɛe a ɛwɔ Rba. Ɛte sɛ nea Sphaeroides RC a wɔde cytochrome c2 (PDB ID: 1L9B) ayɛ biako nso wɔ QB beae a wɔato mu. Nanso, wɔ eyi mu no, N-awie fã a ɛwɔ RC-M polypeptide (a ɛne QB binding site no di nkitaho denam H bond a ɛwɔ Tyr residue a ɛwɔ Q helix no so) gye conformation a ɛnyɛ abɔde mu de, na wɔanhwehwɛ QB conformational nsakrae no mu bio ( 30 ). Nea ɛma awerɛhyem ne sɛ yenhuu saa M polypeptide no deformation yi wɔ RC-LH114-W nhyehyɛe no mu, a ɛkame ayɛ sɛ ɛne N-terminal region a ɛwɔ RC-LH116 RC no yɛ pɛ. Ɛsɛ sɛ yɛhyɛ no nso sɛ bere a wotuu LH1 antenna a egyina detergent so no ase akyi no, wosiesiee apolipoprotein RC ahorow a ɛwɔ PDB no mu no, na eyii quinone atare ne srade a ɛwɔ mu a ɛwɔ RC no ne LH1 ring a atwa ho ahyia no mu ntam no fii hɔ ( 31 , 32 ). RC kɔ so yɛ adwuma efisɛ ɛkora cofactors nyinaa so, gye QB quinone a ɛporɔw, a ɛnyɛ den pii na ɛtaa yera bere a wɔresiesie no (33). Bio nso, wonim sɛ LH1 ne abɔde mu cyclic lipids a woyi fi RC mu no betumi anya nkɛntɛnso wɔ dwumadi ahorow so, te sɛ P+QB-tebea a charge-tetew mu no nkwa nna a ɛyɛ tiaa ( 31 , 34 , 35 ). Enti, yɛsusu sɛ mpɔtam hɔ LH1 ring a atwa RC ho ahyia no a ɛwɔ hɔ no betumi akura QB beae a “wɔato mu” no mu, na ɛnam so akora mpɔtam hɔ nneɛma a atwa yɛn ho ahyia a ɛbɛn QB no so.
Ɛwom sɛ apolipoprotein (a QB quinone nni mu) ne nhyehyɛe a edi mũ no gyina hɔ ma QB beae no dannan ho mfonini abien pɛ de, sen sɛ ɛbɛyɛ nsɛm a esisi toatoa so de, nanso nneɛma bi wɔ hɔ a ɛkyerɛ sɛ wobetumi ayɛ apon a ɛkyekyere no de asiw hydroquinone a ɛsan kyekyere no ano Sɛnea ɛbɛyɛ a wobesiw substrate a wɔbara ano. Ebia nkitahodi a ɛda quinolol ne quinone ntam wɔ baabi a ɛbɛn apolipoprotein QB beae no bɛyɛ soronko, na ɛno ma RC pow no. Wɔakyerɛ bere tenten sɛ nsakrae a ɛba wɔ nipadua no mu no di dwuma wɔ quinones a ɛkyekyere ne nea ɛtew so no mu. Tumi a RC ahorow a wɔahyɛ no nwini wɔ sɛ ɛbɛtew quinones so bere a wɔayɛ nsakrae wɔ sum mu akyi no asɛe ( 36 ); X-ray crystallography kyerɛ sɛ saa ɔsɛe yi fi QB quinones a wɔakyere wɔ “distal” conformation bɛyɛ 4.5 Å fi active proximal position (26) , 37). Yɛhyɛ nyansa sɛ saa distal binding conformation yi yɛ mfinimfini tebea a ɛda apolipoprotein ne ring nhyehyɛe a edi mũ no ntam no ho mfonini, a edi mfiase nkitahodi a ɛne quinone ne QB beae no bue akyi.
Type II RC a wohu wɔ PSII complex a ɛwɔ phototrophic bacteria ne cyanobacteria, algae ne afifide bi mu no wɔ structure ne functional conservation ( 38 ). Nneɛma a wɔahyehyɛ a wɔakyerɛ wɔ Mfonini 6 (D ne E) mu no si nsɛsoɔ a ɛda PSII RCs ne QB beaeɛ a ɛwɔ bacterial RC complex no ntam so dua. Saa ntotoho yi ayɛ nhwɛso a wɔde sua nhyehyɛe ahorow a ɛne quinone kyekyere ne nea ɛtew so a ɛne ne ho di nsɛ kɛse no fi bere tenten. Nhoma ahorow a atwam kyerɛe sɛ conformational nsakrae no ka PSII reduction of quinones ( 39 , 40 ). Enti, sɛ yɛhwɛ RC a wɔkora so wɔ adannandi mu a, ebia saa kwan a ɛkyekyere a kan no na wonhu yi nso betumi ayɛ adwuma wɔ QB beae a PSII RC wɔ wɔ afifide a oxygen wom a ɛma hann mu no nso.
Rps ΔpufW (pufW a wɔpopa a wɔankyerɛw) ne PufW-His (C-terminal 10x His-tagged protein-W a wɔda no adi fi abɔde mu pufW beae) ahorow. palustris CGA009 na wɔkaa ho asɛm wɔ yɛn adwuma a atwam no mu ( 16 ). Wɔsan nyaa saa nkwaboaa yi ne isogenic wuram-su ɔwofo no fii frigye mu denam nkwammoaa kakraa bi a wɔde streaking yɛɛ wɔ PYE (biara yɛ 5 g lita -1) (a wɔde asie LB mu wɔ -80 °C, a 50% ( w/v) glycerol) protein, mmɔkaw a wɔayiyi ne succinate wom) agar [1.5% (w/v)] mprɛte so. Wɔde mprɛte no too sum mu anadwo wɔ dan mu hyew mu wɔ tebea horow a mframa nnim mu, na afei wɔde hann fitaa (~50 μmolm-2 s-1) a OSRAM 116-W halogen bulbs (RS Components, UK) de mae no hyerɛn no nnafua 3 kosi 5 kosii sɛ akuw biako puei. Wɔde colony biako dii dwuma de 10 ml M22+ medium (41) a wɔde 0.1% (w/v) casamino acids (a wɔbɛfrɛ no M22) ahyɛ mu ma. Wɔde amammerɛ no nyin wɔ oxygen tebea a ɛba fam mu wɔ sum mu wɔ 34°C a wɔwosow no 180 rpm nnɔnhwerew 48, na afei wɔde amammerɛ no 70 ml guu mu wɔ tebea koro no ara mu nnɔnhwerew 24. Wɔde semi-aerobic culture a ne dodoɔ yɛ 1 ml di dwuma de inoculate 30 ml M22 medium wɔ 30 ml amansan screw-top transparent glass toa mu na wɔde agitation (~50μmolm-2 s-1) irradiated nnɔnhwerew 48 denam sterile magnetic force Stirring rod. Afei wɔde amammerɛ no bɛyɛ lita 1 guu amammerɛ no mu 30 ml wɔ tebea koro no ara mu, na afei wɔde dii dwuma de hyɛɛ amammerɛ no mu bɛyɛ lita 9 a wɔahyerɛn wɔ ~200 μmolm-2 s-1 mu nnɔnhwerew 72. Wɔnam centrifugation so twaa nkwammoaa no wɔ 7132 RCF simma 30, san de guu ~10 ml 20 mM tris-HCl (pH 8.0) mu, na wɔde siee -20°C kosii sɛ ɛho behiae.
Sɛ woyow wie a, fa deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) ne Roche holoenzyme protease inhibitor tablets abien (Merck, UK) ahwehwɛ bi ka nkwammoaa a wɔasan de ahyɛ mu no ho. Wɔ Fransefo nhyɛso nkwammoaa a ne kɛse yɛ 20,000 psi (Aminco, U.S.A.) mu no, wɔsɛee nkwammoaa no mpɛn 8 kosi 12. Bere a wɔde centrifugation wɔ 18,500 RCF mu simma 15 wɔ 4°C yii nkwammoaa a entumi nsɛee ne nneɛma a entumi nkɔ mu no fii hɔ akyi no, wɔde centrifugation a ɛwɔ 113,000 RCF mu nnɔnhwerew 2 wɔ 43,000°C so maa membrane no fii lysate a wɔde kɔla ayɛ no mu. Tow fã a ɛtumi nwura mu no gu na san fa aduru a ɛwɔ kɔla no gu 20 mM tris-HCl (pH 8.0) ml 100 kosi 200 mu na ma ɛyɛ pɛ kosi sɛ agregates a wotumi hu biara nni hɔ. Wɔde membrane a ɛsensɛn hɔ no too 20 mM tris-HCl (pH 8.0) (Anatrace, USA) a 2% (w/v) β-DDM wom mu dɔnhwerew 1 wɔ sum mu wɔ 4°C a wɔkanyan no brɛoo. Afei centrifuge wɔ 70°C ma 150,000 RCF wɔ 4°C dɔnhwerew 1 na yi nkae a entumi nkɔ mu.
Wɔde solubilizing membrane a efi ΔpufW strain no mu no guu 50 ml DEAE Sepharose ion exchange column a column volumes abiɛsa (CV) a binding buffer [20 mM tris-HCl (pH 8.0) a 0.03% (w / v) β-DDM wom wom mu. Fa CV binding buffers abien hohoro adum no, na afei fa binding buffers abien a 50 mM NaCl wom hohoro adum no. Wɔde linear gradient a ɛyɛ 150 kosi 300 mM NaCl (wɔ binding buffer mu) wɔ 1.75 CV so yii RC-LH116 complex no, na wɔde binding buffer a 300 mM NaCl wom wɔ 0.5 CV so yii binding complex a aka no. Boaboa absorption spectrum a ɛwɔ 250 ne 1000 nm ntam, ma fraction a absorbance ratio (A880/A280) boro 1 wɔ 880 kosi 280 nm, dilute no mprenu wɔ binding buffer no mu, na fa saa kwan koro no ara di dwuma bio wɔ DEAE column no so On purification. Fa afã horow a A880/A280 nsusuwii a ɛkorɔn sen 1.7 ne A880/A805 nsusuwii a ɛkorɔn sen 3.0 no hyɛ mu, yɛ ion nsakrae a ɛto so abiɛsa no, na fa afã horow a A880/A280 nsusuwii a ɛkorɔn sen 2.2 ne A880/A805 nsusuwii a ɛkorɔn sen 5.0 no sie. Wɔde complex a wɔatew ho fã bi no yɛɛ ~2 ml wɔ Amicon 100,000 molecule weight cut-off (MWCO) centrifugal filter (Merck, UK), na wɔde guu Superdex 200 16/600 size exclusion column (GE Healthcare, US) a 200 mM NaCl buffer wom so, na afei wɔde guu buffer koro no ara mu wɔ 1.5 CV. Boaboa absorption spectra a ɛwɔ size exclusion fraction no ano, na fa absorption spectra no a A880/A280 ratios a ɛboro 2.4 ne A880/A805 ratios a ɛboro 5.8 kɔsi 100 A880, na fa di dwuma ntɛm ara ma cryo-TEM grid ahosiesie anaa sie Fa sie -80°C kɔsi sɛ ɛhia.
Wɔde solubilizing membrane a efi PufW-His strain mu no guu 20 ml HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) a 200 mM NaCl ne 0.03% (w/w)) wom mu wɔ IMAC buffer ( GE Healthcare) mu. v) β-DDM a wɔde yɛ adwuma]. Wɔde IMAC buffer CV anum hohoroo adum no, na afei wɔde IMAC buffer CV anum a histidine 10 mM wom hohoroo adum no. Wɔde IMAC buffers anum a histidine 100 mM wom yii core complex no fii adum no mu. Wɔde afã a RC-LH114-W complex no wom no hyɛ ~10 ml mu wɔ tank a wɔakanyan a wɔde Amicon 100,000 MWCO filter (Merck, UK) ahyɛ mu mu, wɔde binding buffer afra mu mpɛn 20, na afei wɔde ka ho akɔ 25 ml Wɔ DEAE Sepharose column no mu no, wodi kan de CV anan a wɔakyekyere wɔ buffer no ho di dwuma. Hohoro adum no ne CV binding buffers anan, afei elute complex no wɔ CVs awotwe so wɔ linear gradient a ɛyɛ 0 kosi 100 mM NaCl (wɔ binding buffer mu), ne CVs anan a aka a ɛwɔ 100 mM binding buffer The residual complexes eluted on the sodium chloride combined with the A880/A280 ratio higher than 2.4 and the Wɔde A880/A805 ratio a ɛboro 4.6 fractions no yɛɛ ~2 ml wɔ Amicon 100,000 MWCO centrifugal filter mu, na wɔde 1.5 CV IMAC dii kan hyɛɛ mu ma Buffer equilibrated Superdex 200 16/600 size exclusion column, na afei wɔde eluted wɔ buffer koro no ara mu wɔ 1.5 CV. Boaboa absorption spectra a ɛwɔ size-exclusion fractions no ano na fa absorption spectra no a A880/A280 ratios a ɛboro 2.1 ne A880/A805 ratios a ɛboro 4.6 kosi 100 A880, a wɔde di dwuma ntɛm ara ma frozen TEM grid ahosiesie anaasɛ wɔde sie -80°C kosi sɛ ɛho behia.
Wɔde Leica EM GP immersion freezer dii dwuma de siesiee TEM grids a ɛyɛ hyew a ɛba fam. Wɔde complex no guu IMAC buffer mu ma ɛbɛyɛɛ A880 a ɛyɛ 50, na afei wɔde 5μl guu QUANTIFOIL 1.2/1.3 carbon-coated copper mesh (Agar Scientific, UK) a wɔde hyerɛn foforo ahyɛ mu. Fa grid no to 20°C ne 60% relative humidity so s 30, afei popa no ma ɛnyow s 3, na afei dum no wɔ nsu ethane mu wɔ -176°C.
Wɔkyerɛw RC-LH114-W complex no ho nsɛm wɔ eBIC (Electronic Bioimaging Center) (British Diamond Light Source) so denam Titan Krios microscope a ɛyɛ adwuma wɔ ahoɔhare ahoɔden a ɛyɛ 300kV, a ɛwɔ din mu nkɔanim a ɛyɛ 130,000× ne ahoɔden a ɛyɛ- Paw ɔkwan a ɛyɛ 20 eV. Wɔde Gatan 968 GIF Quantum a ɛwɔ K2 peak detector dii dwuma de kyerɛw mfonini ahorow wɔ akontaabu kwan so de boaboaa nsɛm ano. Pixel a wɔasusuw ho no kɛse yɛ 1.048Å, na dodow a wɔde ma no yɛ 3.83 e-Å-2s-1. Ɔboaboaa sini no ano wɔ sikani 11 mu na ɔkyekyɛɛ mu yɛɛ no ​​afã 40. Fa beae a wɔde carbon akata so no san hwɛ microscope no so, na afei boaboa sini abiɛsa ano wɔ tokuru biara mu. Sɛ wɔka ne nyinaa bom a, wɔboaboaa sini ahorow 3130 ano, a defocus values ​​wɔ -1 ne -3μm ntam.
Wɔde afiri a wɔde hwɛ nneɛma nketenkete koro no ara na ɛboaboaa RC-LH116 complex no ho nsɛm ano wɔ Asterbury Biostructure Laboratory (University of Leeds, UK). Wɔboaboaa nsɛm no ano wɔ akontaabu kwan so a wɔma ɛyɛ kɛse 130 k, na wɔde piksel kɛse no yɛɛ 1.065 Å a wɔde dodow a ɛyɛ 4.6 e-Å-2s-1. Wɔde sikani 12 na ɛkyeree sini no na wɔkyekyɛɛ mu yɛɛ no ​​afã 48. Sɛ wɔka ne nyinaa bom a, wɔboaboaa sini ahorow 3359 ano, a defocus values ​​wɔ -1 ne -3μm ntam.
Wɔyɛ data dwumadie nyinaa wɔ Relion 3.0 pipeline (42) mu. Fa Motioncorr 2 (43) siesie beam no kankan denam dose weighting so, na afei fa CTFFIND 4.1 (44) kyerɛ CTF (contrast transfer function) parameter. Mfonini nketenkete a wɔtaa yɛ wɔ saa mfitiaseɛ dwumadie akwan yi akyi no wɔakyerɛ wɔ Mfonini 2. S16. Wɔnam nsa a wɔde paw bɛyɛ piksel 250 a ɛyɛ 1000 a ɛwɔ 250-pixel frame mu na reference two-dimensional (2D) classification biara nni mu no so na ɛyɛ automatic selection template no, na ɛnam so pow saa nkyekyɛmu a ɛne sample efĩ hyia anaasɛ enni su biara a wotumi hu. Afei, wɔyɛɛ mfonini nketewa no nyinaa a wɔpaw no ankasa, na na RC-LH114-W no yɛ nneɛma nketenkete 849,359, na na RC-LH116 a ɛyɛ den no yɛ nneɛma nketenkete 476,547. Nneɛma nketenkete a wɔapaw no nyinaa afa 2D nkyekyɛmu a ɛnyɛ nhwɛsoɔ mu mprenu, na wɔ mmirikatuo biara akyi no, wɔpo nneɛma nketenkete a ɛhyia carbon beaeɛ, nhwɛsoɔ efĩ, nneɛma a ɛda adi pefee anaa nneɛma nketenkete a ɛka bom denneennen, na ɛde 772,033 (90.9%) ne 359,678 (75.5%) ) Wɔde nneɛma nketenkete di dwuma ma 3D nkyekyɛmu a RC-LH114-W ne RC-LH116 sɛnea ɛte biara. Wɔde stochastic gradient descent kwan no na ɛyɛɛ mfitiaseɛ 3D nkyerɛkyerɛmu nhwɛsoɔ no. Sɛ wɔde mfitiaseɛ nhwɛsoɔ no di dwuma sɛ nhwɛsoɔ a, wɔkyekyɛ nneɛma nketenkete a wɔapaw no mu akuo anan wɔ 3D mu. Fa model a ɛwɔ saa ɔfa yi mu di dwuma sɛ nhwɛsoɔ, yɛ 3D refining wɔ particles a ɛwɔ category kɛseɛ no mu, afei fa mfitiaseɛ 15Å low-pass filter no kata solvent beaeɛ no so, fa piksel 6 a ɛyɛ soft edges ka ho, na post-process piksel no de siesie Gatan K2 peak Modulation transfer function a ɛwɔ top detector no mu. Wɔ RC-LH114-W dataset no fam no, wɔsesaa saa mfitiaseɛ nhwɛsoɔ yi denam density a ɛyɛ den a ɛwɔ mask no anoano (a wɔatwa afiri core complex density a ɛwɔ UCSF Chimera mu) no a wɔyii no so. Wɔde nhwɛsoɔ a ɛfiri mu baeɛ (RC-LH114-W ne RC-LH116 nsusuiɛ yɛ 3.91 ne 4.16 Å, sɛdeɛ ɛteɛ biara) di dwuma sɛ nhwɛsoɔ ma 3D nkyekyɛmu a ɛtɔ so mmienu. Wɔakyekyɛ nneɛma nketenkete a wɔde di dwuma no mu akuwakuw ayɛ no 3D kuw a edi kan no na enni abusuabɔ a emu yɛ den a ɛne mpɔtam hɔfo wɔ. Nneɛma a ɛka bom anaasɛ enni nhyehyɛe mu nneɛma a ɛda adi pefee. Wɔ 3D nkyekyɛmu a ɛtɔ so mmienu akyi no, wɔpaw ɔfa a ɛwɔ nsusuwii a ɛkorɔn sen biara [Wɔ RC-LH114-W fam no, ɔfa biako yɛ nneɛma nketenkete 377,703 (44.5%), wɔ RC-LH116 fam no, akuw abien na ɛwɔ hɔ, a ne nyinaa yɛ abɔde nketenkete 260,752 (54.7%) , Faako a wɔyɛ pɛ bere a wɔahyehyɛ no pɛpɛɛpɛ wɔ mfitiase kyinhyia no akyi nkutoo a nsonsonoe ketewaa bi wom Wɔsan yi nneɛma nketenkete a wɔapaw no wɔ adaka a ne kɛse yɛ piksel 400 mu na wɔde 3D a wɔde siesie no tew ho. Wɔde mfitiaseɛ 15Å low-pass filter, 3 pixel map ntrɛmu ne 3 pixel soft mask na ɛyɛ solvent mask no. Sɛ wɔde CTF a wɔde yɛ nneɛma nketenkete biara di dwuma, nteɛso a wɔde yɛ nneɛma nketenkete biara kankan ne nea wɔde yɛ CTF a wɔde yɛ nneɛma nketenkete biara a ɛto so abien a, wɔyɛ 3D ahotew, solvent masking ne post-processing wɔ anammɔn biara akyi de siesie texture a efi mu ba no bio. Sɛ yɛde FSC (Fourier Shell Correlation Coefficient) cut-off value a ɛyɛ 0.143 di dwuma a, RC-LH114-W ne RC-LH116 nhwɛsoɔ a ɛtwa toɔ no nsusuiɛ yɛ 2.65 ne 2.80Å, sɛdeɛ ɛteɛ biara. FSC curve a ɛwɔ model a etwa toɔ no mu no wɔakyerɛ wɔ Mfonini 2. S17.
Wɔtwe protein ahorow a ɛtoatoa so nyinaa fi UniProtKB: LH1-β (PufB; UniProt ID: Q6N9L5); LH1-α (PufA; UniProtID: Q6N9L4); RC-L (PufL; UniProt ID: O83005); RC-M (PufM; UniProt ID: A0A4Z7); RC-H (PuhA; UniProt ID: A0A4Z9); Protein-W (PufW; UniProt ID: Q6N1K3) a ɛwɔ nipadua no mu. Wɔde SWISS-MODEL (45) yɛɛ homology model a ɛfa RC ho, a ɛwɔ protein ntoatoaso a ɛwɔ RC-L, RC-M ne RC-H ne Rba ahwehwɛ nhyehyɛe. sphaeroides RC na wɔde dii dwuma sɛ nhwɛsode (PDB ID: 5LSE) ( 46 ). Fa “fit map” adwinnade a ɛwɔ UCSF Chimera mu no fata model a wɔayɛ no ma ɛne map no hyia (47), ma protein nhyehyɛe no tu mpɔn, na cofactor [4×BChl a (monomer nhomakorabea nkae din = BCL), 2×BPh a (BPH), UQ10 ahorow biako anaa abien (U10), dade biako a ɛnyɛ heme (Fe) ne 3,4-dihydrohexacarbonylcholine biako (QAK)] de Coot (48) di dwuma de ka ho. Esiane sɛ QAK nni monomer nhomakorabea nti, wɔde eLBOW adwinnade a ɛwɔ PHENIX (49) mu no yɛɛ parameterized.
Afei, wɔyɛɛ LH1 subunit no. Mfiase no, wɔde adwinnade a wɔde si dan a ɛyɛ adwuma ankasa wɔ PHENIX (49) mu no dii dwuma de yɛɛ LH1 ntoatoaso no fã bi a wɔde asase mfonini no ne LH1-α ne LH1-β protein ntoatoaso no dii dwuma sɛ nsɛm a wɔde hyɛ mu. Paw LH1 subunit a edi mũ sen biara, yi na fa gu Coot mu, fa nsa ka ntoatoaso a ɛyera no ho wɔ mu, na fa nsa siesie nhyehyɛe no nyinaa ansa na wode BCls abien a (BCL) ne spirilloxanthin (CRT) aka ho [sɛnea Rps a ɛfa ho kyerɛ no LH1 complex no density ne carotenoid a wonim no. Nnomaa ahorow (17)]. Kɔpi LH1 subunit a edi mũ no, na fa UCSF Chimera “Docking Map Tool” no di dwuma de dock wɔ beae a ɛbɛn a ɛnyɛ model a LH1 density no, na afei siesie no yiye wɔ Coot mu; san yɛ adeyɛ no bio kosi sɛ wɔbɛyɛ LH1 subunits nyinaa ho nhwɛso. Wɔ RC-LH114-W nhyehyeɛ no ho no, ɛnam sɛ wɔyi density a wɔankyekyɛ wɔ Coot no mu no so no, wɔkyekyɛ protein no mu firi nneɛma a ɛnyɛ protein a aka wɔ USCF Chimera map no mu na wɔde Autobuild adwinnadeɛ no si mfitiaseɛ nhwɛsoɔ no so, ne subunits a aka (protein-W) Modeling. Wɔ PHENIX (49) mu no. Fa ntoatoaso biara a ɛyera ka nhwɛso a efi mu ba no ho wɔ Coot (48) mu, na afei fa nsa siesie subunit no nyinaa. Density a aka a wɔankyekyɛ no fata lipids (PDB monomer nhomakorabea ID a ɛyɛ CDL = CDL, POPC = 6PL ne POPG = PGT), β-DDM detergent (LMT) ne UQ10 molecules (U10) a wɔaka abom. Fa PHENIX optimization (49) ne manual optimization wɔ Coot (48) mu di dwuma de yɛ mfitiaseɛ nhwɛsoɔ a ɛdi mũ no pɛpɛɛpɛ kɔsi sɛ model akontabuo ne aniwa su a ɛwɔ fit no mu no rentumi nkɔ anim bio. Awiei koraa no, fa LocScale (50) di dwuma de yɛ mpɔtam hɔ asase mfonini no nnam, na afei yɛ kyinhyia afoforo pii a ɛyɛ density a wɔankyekyɛ no ho nhwɛso ne automatic ne manual optimization.
Peptide ahorow, cofactors ne lipids ne quinones afoforo a wɔde ahyɛ wɔn densities mu no wɔakyerɛ wɔ Mfonini 1 ne 2. S18 kosi S23. Akontaabuo ho nsɛm a ɛfa nhwɛsoɔ a ɛtwa toɔ no ho no wɔakyerɛ wɔ Table S1 mu.
Gye sɛ wɔakyerɛ sɛ ɛnte saa, wɔboaboaa UV/Vis/NIR absorption spectra ano wɔ Cary60 spectrophotometer (Agilent, USA) so wɔ 1 nm ntam fi 250 nm kosi 1000 nm ne nkabom bere a ɛyɛ 0.1s.
Fa nhwɛsoɔ no hyɛ quartz cuvette a ɛwɔ mm 2 kwan kɔ A880 a ɛyɛ 1 mu, na boaboa absorption spectrum no ano wɔ 400 ne 1000 nm ntam. Wɔboaboaa dichroic spectra kurukuruwa no ano wɔ Jasco 810 spectropolarimeter (Jasco, Japan) so wɔ 1 nm ntam wɔ 400 nm ne 950 nm ntam wɔ scan rate a ɛyɛ 20 nm min-1.
Wɔnam core complex no a wɔma ɛyɛ A880 bɛyɛ 50. Fa 10μl volume no hyɛ 990μl binding buffer anaa methanol mu, na boaboa absorption spectrum no ano ntɛm ara na ama BChl asɛe no ayɛ ketewaa. Wɔde extinction coefficient a ɛwɔ 771 nm a ɛyɛ 54.8 mM-1 cm-1 na ɛbuu BChl a ɛwɔ methanol nhwɛsoɔ biara mu, na wɔkyerɛɛ extinction coefficient ( 51 ). Kyekyɛ BChl dodoɔ a wɔasusu no mu 32 (RC-LH114-W) anaa 36 (RC-LH116) de kyerɛ core complex dodoɔ, a afei wɔde kyerɛ absorption spectrum a ɛwɔ nhwɛsoɔ korɔ no ara a wɔaboaboa ano wɔ buffer Extinction coefficient no mu. sa so. Wɔsusuu mprɛnsa mpɛn pii maa nhwɛsoɔ biara, na wɔde BChl Qy maximum no absorbance a wɔkyekyɛ mu no dii dwuma de buu akontaa. RC-LH114-W a wɔsusuu wɔ 878 nm no asetɔreɛ nsusuiɛ yɛ 3280±140 mM-1 cm-1, berɛ a RC-LH116 a wɔsusuu wɔ 880 nm no asetɔreɛ nsusuiɛ yɛ 3800±30 mM-1 cm-1.
Wɔkyerɛɛ UQ10 dodow sɛnea ɔkwan a ɛwɔ (52) mu no te. Ne tiawa mu no, wɔde Agilent 1200 HPLC nhyehyɛe no yɛɛ reverse phase HPLC (RP-HPLC). Fa RC-LH116 anaa RC-LH114-W bɛyɛ 0.02 nmol gu 50μl 50:50 methanol:chloroform a 0.02% (w/v) ferric chloride wom mu, na fa Beckman Coulter Ultrasphere ODS 4.6 mm a wɔadi kan ayɛ no pɛpɛɛpɛ no gu mu wɔ 1 ml-1 min-1 mu wɔ 40°C wɔ HPLC mu aduru a wɔde yɛ aduru (80:20 methanol:2-propanol) wɔ ×25 cm adum so. Yɛ isocratic elution wɔ HPLC solvent mu de hwɛ absorbance wɔ 275 nm (UQ10), 450 nm (carotenoids) ne 780 nm (BChl) dɔnhwerew 1. Wɔde peak a ɛwɔ 275 nm chromatogram no mu wɔ simma 25.5 mu no boom, a na nnuru foforo biara a wotumi hu nni mu. Wɔde beaeɛ a wɔaka abom no di dwuma de bu molar dodoɔ a UQ10 a wɔayi afiri mu no de gyina calibration curve a wɔbuu ano firi injection of pure standards firi 0 kɔsi 5.8 nmol (Mfonini S14). Wɔyɛɛ nhwɛsode biara mu nhwehwɛmu wɔ mprɛnsa mu, na mfomso a wɔbɔɔ ho amanneɛ no ne SD a ɛwɔ sɛ wɔkyekyem pɛpɛɛpɛ no hyia.
Wɔde 30 μM a wɔatew apɔnkɔ koma cytochrome c2 (Merck, UK) ne 0 kosi 50 μMUQ2 (Merck, UK) na ɛyɛɛ ano aduru a RC-LH1 complex no wom a Qy a ɛtwetwe kɛse yɛ 0.1. Wɔyɛɛ 1-ml nhwɛsode abiɛsa wɔ UQ2 dodow biara mu na wɔde too sum mu anadwo wɔ 4°C mu sɛnea ɛbɛyɛ a wɔbɛhwɛ ahu sɛ wɔbɛdannan wɔn ho koraa akɔ sum no mu ansa na wɔasusuw. Wɔde ano aduru no guu OLIS RSM1000 modular spectrophotometer a wɔde 300 nm ogya/500 line grating, 1.24 mm inlet, 0.12 mm mfinimfini ne 0.6 mm outlet slits ahyɛ mu. Wɔde 600 nm tenten pass filter si sample phototube no ano ne reference photomultiplier tube no ano na ama excitation hann no afi mu. Wɔhwɛɛ absorbance no wɔ 550 nm a bere a wɔde ka bom yɛ 0.15 s. Anigye hann no fi 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) nam fiber optic cable a ano yɛ den 90% denam DC2200 controller (Thorlabs Ltd., UK) so na ɛba hann fibea no so wɔ anim a ɛyɛ 90° of. Wɔde susudua no ne ahwehwɛ no bɔ abira na ama hann biara a mfiase no na nhwɛsode no annye no asan aba. Hwɛ absorbance 10 s ansa na hann a ɛyɛ 50 s. Afei wɔsan hwɛɛ absorbance no so 60 s wɔ sum mu de hwɛɛ baabi a quinolol fi ne ho so tew cytochrome c23 + so (hwɛ Mfonini S8 ma raw data).
Wɔnam linear initial rate a wɔde sii hɔ wɔ 0.5 kɔsi 10 s mu (egyina UQ2 dodoɔ so) ne nhwɛsoɔ mmiɛnsa no nyinaa dodoɔ a wɔde yɛɛ nkyɛmu wɔ UQ2 dodoɔ biara mu no so yɛɛ data no ho adwuma. Wɔde RC-LH1 dodoɔ a wɔde extinction coefficient a ɛfa ho no bu akontaa no dii dwuma de danee rate no kɔɔ catalytic efficiency mu, a wɔayɛ ho mfonini wɔ Origin Pro 2019 (OriginLab, USA), na wɔde ahyɛ Michaelis-Menten model no mu de kyerɛɛ Km ne Kcat botaeɛ a ɛda adi.
Wɔ bere tiaa mu absorption susudua mu no, wɔde RC-LH1 nhwɛsode no yɛɛ ~2μM wɔ IMAC buffer a 50 mM sodium ascorbate (Merck, USA) ne 0.4 mM Terbutin (Merck, USA) wom mu. Wɔde ascorbic acid di dwuma sɛ afɔrebɔ ɛlɛtrɔnik a wɔde ma, na wɔde tert-butaclofen di dwuma sɛ QB inhibitor de hwɛ hu sɛ RC a wɔde ma titiriw no kɔ so tew (kyerɛ sɛ, ɛnyɛ photooxidized) wɔ susuw nhyehyɛe no nyinaa mu. Bɛyɛ 3 ml nhwɛsode na wɔde ka amanne kwan so rotating cell (bɛyɛ 0.1 m wɔ ne kɛse, 350 RPM) a 2 mm optical kwan tenten de hwɛ hu sɛ nhwɛsode a ɛwɔ laser kwan no mu no wɔ bere a ɛdɔɔso ma esum adaptation ntam excitation pulses. Fa ~100-fs laser pulses ma Ti: Sapphire laser nhyehyɛe (Spectra Physics, USA) no yɛ kɛse de kanyan nhwɛsode no wɔ 880 nm wɔ repetition rate a ɛyɛ 1 kHz (20 nJ ma NIR anaa 100 nJ ma Vis). Ansa na wobɛboaboa nsɛm ano no, fa nhwɛsode no to hann a ɛkanyan adwene mu bɛyɛ simma 30. Sɛ wɔde wɔn ho hyɛ mu a, ɛbɛma QA ayɛ adwuma (ebia ɛbɛtew QA so pɛnkoro anaa mprenu). Nanso yɛsrɛ sɛ hyɛ no nsow sɛ saa adeyɛ yi yɛ nea wotumi dannan efisɛ bere tenten a wɔde yɛ nsakrae wɔ sum mu akyi no, RC bɛsan de nkakrankakra akɔ QA dwumadi mu. Wɔde Helios spectrometer (Ultrafast Systems, U.S.A.) dii dwuma de susuw bere tiaa mu spectro a bere a ɛkyɛ yɛ -10 kosi 7000 ps . Fa Surface Xplorer software (Ultrafast Systems, USA) yiyi data ahorow no mu, afei ka bom na yɛ no pɛpɛɛpɛ. Fa CarpetView software package (Light Conversion Ltd., Lithuania) di dwuma de fa data set a wɔaka abom no di dwuma de nya differential spectra a ɛfa decay ho, anaasɛ fa function a convolves exponents pii ne adwinnade mmuae ma ɛne single-wavelength spectral evolution wɔ Origin (OriginLab, USA) hyia di dwuma.
Sɛnea yɛaka wɔ atifi hɔ (53) no, wɔyɛɛ photosynthetic film a LH1 complex a enni RC ne peripheral LH2 antenna nyinaa wom. Wɔde membrane no guu 20 mM tris (pH 8.0) mu na afei wɔde guu quartz cuvette a ɛwɔ 2 mm optical kwan mu. Wɔde 30nJ laser pulse dii dwuma de kanyan nhwɛsode no wɔ 540 nm a bere a ɛkyɛ a ɛyɛ -10 kosi 7000 ps. Di data set no ho dwuma sɛnea wɔakyerɛkyerɛ mu ama Rps. pal nhwɛsode.
Wɔnam centrifugation so yɛɛ membrane no pelleted wɔ 150,000 RCF nnɔnhwerew 2 wɔ 4°C, na afei wɔsan de ne absorbance wɔ 880 nm guu 20 mM tris-HCl (pH 8.0) ne 200 mM NaCl mu. Fa membrane no gu denam 2% (w/v) β-DDM a wode bɛkanyan no nkakrankakra dɔnhwerew 1 wɔ sum mu wɔ 4°C mu no so. Wɔde nhwɛsode no guu 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) mu ma protein dodow a ɛyɛ 2.5 mg ml-1 (Bio-Rad nhwehwɛmu). Wɔyɛɛ adwuma foforo fii ɔkwan a wɔadi kan atintim no so ( 54 ), na wofii ase de protein 50 μg a wɔde guu mu yɛɛ no ​​TEAB a ne nyinaa yɛ 50 μl a sodium laurate 1% (w/v) wom (Merck, UK). Afei sonication ma 60 s, wɔtew so ne 5 mM tris (2-carboxyethyl) phosphine (Merck, UK) wɔ 37 ° C simma 30. Sɛ wopɛ S-alkylation a, fa nhwɛsode no to 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) mu na fa fi 200 mM isopropanol stock solution mu simma 10 wɔ dan mu hyew mu. Wɔyɛɛ proteolytic digestion denam 2 μg trypsin/endoproteinase Lys-C afrafra (Promega UK) a wɔde kaa ho na wɔde too 37°C nnɔnhwerew 3 so. Wɔnam ethyl acetate 50 μl ne LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) a wɔde 10 μl 10% (v/v) kaa ho na wɔde vortex yɛɛ no ​​60 s so yii laurate surfactant no. Wɔnam centrifugation so wɔ 15,700 RCF simma 5 na ɛhyɛɛ phase mpaapaemu no ho nkuran. Sɛnea nea ɔyɛe no nhyehyɛe kyerɛ no, wɔde C18 spin column (Thermo Fisher Scientific, UK) dii dwuma de ahwɛyiye twetwee na wɔde nkyene fii mu wɔ ɔfã a ɛwɔ fam a peptide no wom no mu. Bere a wɔde vacuum centrifugation ayow akyi no, wɔde nhwɛsode no guu 0.5% TFA ne 3% acetonitrile mu, na wɔde nanoflow RP chromatography a wɔde mass spectrometry ka ho de nhyehyɛe no parameters a wɔadi kan aka ho asɛm kɔ akyiri no yɛɛ 500 ng mu nhwehwɛmu.
Fa MaxQuant v.1.5.3.30 (56) di dwuma ma protein a wɔde hu ne dodow de hwehwɛ Rps. palustris proteome ho nsɛm a wɔkora so (www.uniprot.org/proteomes/UP000001426). Wɔde mass spectrometry proteomics data no akɔto ProteomeXchange Alliance no mu denam PRIDE ahokafoɔ akoraeɛ (http://proteomecentral.proteomexchange.org) wɔ dataset identifier PXD020402 ase.
Sɛ wɔde RPLC a wɔde electrospray ionization mass spectrometry aka ho nhwehwɛmu a, wɔsiesiee RC-LH1 complex no fii wuram-su Rps mu. Sɛ yɛde ɔkwan a wɔadi kan atintim (16) di dwuma a, protein dodow a wɔyɛe wɔ palustris nkwammoaa mu no yɛ 2 mg ml-1 wɔ 20 mM Hepes (pH 7.8), 100 mM NaCl ne 0.03% (w/v) β- (Bio-Rad nhwehwɛmu) ) DDM mu. Sɛnea nea ɔyɛe no nhyehyɛe kyerɛ no, fa 2D purification kit (GE Healthcare, USA) yi protein 10 μg denam osutɔ kwan so, na fa nsu a ɛtɔ gu fam no gu 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile ne 20% (v/v) nsu mu. Wɔde RPLC (Dionex RSLC) a wɔde mass spectrometry (Maxis UHR-TOF, Bruker) aka ho yɛɛ microliter anum mu nhwehwɛmu. Fa MabPac 1.2×100 mm adum (Thermo Fisher Scientific, UK) di dwuma ma mpaapaemu wɔ 60°C ne 100μlmin -1, a ɛwɔ gradient a ɛyɛ 85% (v / v) aduru A [0.1% (v / v) FA ne 0.02% (V/v) TFA nsuo mu aduru] kɔ 85%(v/v) aduru B [0.1%(v/v) FA ne 0.02%(v/v) wɔ 90%(v/v) acetonitrile TFA mu] Sɛ wɔde standard electrospray ionization source ne default parameters di dwuma bɛboro simma 60 a, mass spectrometer no nya 100 kosi 2750 m/z (mass-to-charge ratio). Ɛnam ExPASy bioinformatics resource portal FindPept adwinnadeɛ (https://web.expasy.org/findpept/) mmoa so no, yɛ mass spectrum no map kɔ subunits a ɛwɔ complex no mu.
Wɔde nkwammoaa no nyin nnɔnhwerew 72 wɔ 100 ml NF-low (10μMm-2 s-1), medium (30μMm-2 s-1) anaa high (300μMm-2 s-1) hann ase. M22 aduru (M22 aduru a wɔayi ammonium sulfate afi mu na wɔde sodium acetate asi sodium succinate ananmu) wɔ 100 ml toa a wɔde nkɔnsɔnkɔnsɔn ayɛ mu (23). Wɔ 30-s kyinhyia anum mu no, wɔde 0.1 micron ahwehwɛ nhwiren yɛɛ nhwiren a ne kɛse yɛ 1:1 de lyse nkwammoaa no na wɔde yɛɛ nwini wɔ nsukyenee so simma 5. Wɔde centrifugation wɔ 16,000 RCF simma 10 wɔ benchtop microcentrifuge mu yii ade a entumi nkɔ mu, nkwammoaa a entumi nsɛee ne ahwehwɛ nhwiren no fii hɔ. Wɔtetew membrane no mu wɔ Ti 70.1 rotor a RCF 100,000 wɔ 20 mM tris-HCl (pH 8.0) a sucrose gradient 40/15% (w/w) wom mu nnɔnhwerew 10.
Sɛnea yɛaka ho asɛm wɔ yɛn adwuma a atwam no mu no, immunodetection of the His tag on PufW (16). Ne tiawa mu no, core complex a wɔatew ho (11.8 nM) anaasɛ membrane a RC dodow koro no ara wom (a wɔde oxidation yi nsonsonoe spectrum a wɔatew so no fi mu na ɛne adesoa a ɛwɔ stained gel no so no hyia) wɔ 2x SDS loading buffer (Merck, UK) mu a wɔde ahyɛ mu mprenu. Wɔtetew protein ahorow no mu wɔ 12% bis-tris NuPage gel a wɔayɛ no sɛnea ɛte (Thermo Fisher Scientific, UK) so. Wɔde Coomassie Brilliant Blue (Bio-Rad, UK) guu gel bi so de hyɛɛ RC-L subunit no mu na wɔayɛ ho mfonini wɔ wɔn adwene mu. Wɔde protein a ɛwɔ gel a ɛto so abien no so no guu polyvinylidene fluoride (PVDF) a wɔde methanol ayɛ mu adwuma (Thermo Fisher Scientific, UK) mu de yɛɛ immunoassay. Wɔde PVDF membrane no siw ano wɔ 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 ne 5% (w / v) nufusu a wɔayam no mu, na afei wɔde anti-His primary antibody (wɔ Dilute the antibody buffer [50 mM tris-HCl (pH 7.6) mu no too mu, na wɔde too mu, na wɔde ahyɛ mu 150 mM NaCl ne 0.05% (v/v) Tween-20] wɔ 1:1000 A190-114A, Bethyl Laboratories, U.S.A.) mu nnɔnhwerew 4. Bere a wɔahohoro mpɛn 3 simma 5 wɔ antibody buffer mu akyi no, wɔde horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (a wɔafrafra mu 1:10,000 wɔ antibody buffer mu) kaa membrane no ho (Cyanagen, Italy) ne Amersham Imager 600 (GE Akwahosan Dwumadibea, UK).
Ɛdenam ahoɔden kyekyɛ a wɔbɛtwetwe wɔ stained gel anaa immunoassay lane biara mu, a wɔde beae a ɛwɔ peak no ase no abom na wɔabu RC-L (stained gel) ne Protein-W (immunoassay) ahoɔden ratio, wɔ ImageJ (57) Process the image. Wɔdanee saa nsusuwii ahorow yi yɛɛ no ​​molar nsusuwii denam fa a wɔfaa no sɛ RC-L ne protein-W nsusuwii a ɛwɔ RC-LH114-W nhwɛsode a ɛho tew no mu yɛ 1:1 na wɔmaa data a wɔahyehyɛ no nyinaa yɛɛ nea ɛfata sɛnea ɛfata no so.
Sɛ wopɛ nneɛma foforo a wode bɛka saa asɛm yi ho a, yɛsrɛ wo hwɛ http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
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David J. K. Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Okuafo, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe , Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Hann afiri 1 complex a ɛwɔ reaction center no nhyehyɛe a ɛkorɔn no ma yenya nhumu foforo wɔ quinone dynamics no ho.
David J. K. Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Okuafo, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe , Mark J. Dickman, Dewey Holten, Christine Kirmaier, Andrew Hitchcock, C. Neil Hunter
Hann afiri 1 complex a ɛwɔ reaction center no nhyehyɛe a ɛkorɔn no ma yenya nhumu foforo wɔ quinone dynamics no ho.
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