Azịza ngwa ngwa
An eight-residue peptide — Ac-VCTLCSCY — was synthesized from the conserved metal-binding motif of cobalt nitrile hydratase, loaded with one equivalent of Co(I…
Nsogbu Enzyme Ezubere Peptide ka ọ gafere
Acetyl Hexapeptide 1 N'ogbe Isi mmalite Peptide The Co-ụdị nitrile hydratase nọ n'ọrụ saịtị na-ejide a obere-spin Co(III) na N2S3 ngalaba nhazi mbụ: Nitrogen amidate abụọ sitere na ọkpụkpụ azụ na sulfur atọ sitere na cysteine na steeti oxidation atọ dị iche iche - thiolate., sulfate na sulfate. Nke 1.8 Å kristal Ọdịdị nke Pseudonocardia thermophila enzyme (PDB 1IRE) na-edozi αCys111 dị ka cysteine-sulfinic acid na αCys113 dị ka cysteine-sulfenic acid., otu ụkpụrụ ahụ a na-ahụ na enzyme iron.
Ịnweta ebe ahụ abụghị nzọụkwụ mpịaji na-akpaghị aka. Maturation chọrọ protein activator, na maka PtNHase Zhou na ndị ọrụ ibe ha tụpụtara usoro mgbanwe α-subunit: Apo-α2β2 tetramer na-etolite na mbụ, otu α-subunit binds two Co(II)-nwere protein, A na-etinye cobalt na metal gbakwunyere equatorial cysteine na-agbaze, na α-subunits na-arụ ọrụ ugbu a na-atụgharị n'ime apo-tetramer. Ọmụmụ ihe ndị Frontiers na-ekwu n'ụzọ doro anya na amatachaghị etu nke a si eme na ọkwa atọm.
Nke ahụ bụ nsogbu nkọwa. Na holoenzyme, ntinye ígwè, cysteine oxidation, Mgbakọ subunit na nsonye activator na-eme n'otu ebe n'oge ndị na-agafe agafe. Enweghị ike ịkenye mgbanwe spectroscopic na ihe nlele enzyme tozuru oke na otu n'ime ihe ndị ahụ, n'ihi na ndị ọzọ ka na-agba ọsọ. Ịwepụ ihe mkpofu anaghị ewepụ mkpa ọ dị; ọ na-ewepụ ihe mgbagwoju anya nke na-eme ka usoro obodo ghara ịgụta otu mgbanwe n'otu oge.
Ihe "Modeled on the Motif" na-akọwapụta n'ezie
Ebumnuche echekwara bụ CXXCSCX, na ọmụmụ na-akọwa ya dị ka ebe nile n'etiti NHases. Ihe owuwu ahụ sitere na PtNHase motif bụ Ac-VCTLCSCY: fọduru asatọ, molekul uka 933.13 Na, ya na otu acetyl na N-terminus agbakwunyere iji kwalite solubility. Enyere peptide ahụ ka ọ bụrụ ntụ ntụ lyophilized nke >98.5% ịdị ọcha ma jiri ya mee ihe.
Akụkụ ọnọdụ ọ bụla na-arụ ọrụ. Oghere cysteine na-edobe ebe ndị thiolates na-anọdụ ala n'ebe ibe ha nọ na n'elu ígwè. Ser6 na-anọdụ n'etiti nhazi nke cysteine , na n'ụdị mgbakọ na mwepụ akụkụ ya hydrogen-bonds iji kechie cysteine sulfur site na mmiri abụọ doro anya nke dị n'èzí.. C-terminal tyrosine dị na peptide nnwale mana etinyere ya na otu methyl na ụdị DFT., dị ka agbụ akụkụ nke Val na-adịghị ahazi, Thr na Leu; N-terminal acetyl ka emere ka ọ bụrụ formyl. Ihe fọrọ nke nta ka ọ bụrụ atọm ọkpụkpụ azụ niile gbakwunyere agbụ akụkụ nke Cys2, Cys5, Ser6 na Cys7 ka edobere.
Oke eziokwu nke ihe nrụpụta bụ mgbanwe. Otu peptide fọdụrụnụ asatọ na nchekwa anaghị ejide otu geometry nhazi; ọ na-egosi ọtụtụ ihe, nke bụ kpọmkwem ihe mere EPR ụdịdị dị iche iche na-egosi abụọ spin state karịa otu. A na-agbajikwa ụdị ndị ahụ ma jiri ihe na-aga n'ihu na-edozi ya (e = 80.4) gbakwunyere mmiri doro anya abụọ - ọgwụgwọ ezi uche dị na ya, ma ọ bụghị peptide zuru ezu.
Nsonaazụ nkọwapụta bụ kpọmkwem. E wepụrụ okpu acetyl, ihe fọdụrụ edochiri, ma ọ bụ usoro cysteine na-agbanwe agbanwe na-agbanwe ntọala ligand. Enweghị spectroscopy nke ala na-enwetaghachi nnwale echere na peptide nwere usoro ligand na-ezighi ezi; ọ ga-akọ naanị maka ihe dị iche iche nwere otu oke oke.
Ihe kpatara Peptide dị mkpụmkpụ ji bụrụ ihe nlere anya nke a na-achịkwa, Ọ bụghị Enzyme dị mfe
Ajụjụ ndị odee jụrụ dị warara: Usoro NHase metal-binding nke echekwara n'onwe ya nwere ozi kemịkalụ zuru oke iji bind Co(II) na ime mgbanwe na etiti metal na peptide mgbe ikpughe dioxygen? Nke ahụ bụ ajụjụ gbasara usoro, na usoro bụ ihe peptide sịntetik na-ewepụta na-enweghị protein ndị ọzọ.
Ejirilarị otu ikpo okwu ahụ gafee ọla. Otu peptide ahụ na-ejikọta Fee dị elu(II) etiti anaerobically, na O₂ ikpughe na mbụ na-ebute n'etiti-spin (S = 3/2) Fe(III) etiti nke na-atụgharị gaa na obere ogho Fe(III) dị ka equatorial cysteines oxidize. Ihe owuwu nke enwere ike ibu ígwè wee jiri cobalt, ma na-agụ site na otu spectroscopies, bụ ihe ndina nyochagharị ọzọ karịa molecule na-apụ apụ.
Ntụnyere mpụga kacha nso bụ SODA peptide (ACDLPCG), usoro nke fọdụrụ asaa sitere na nickel superoxide dismutase. N'ime Ịmụ akwụkwọ na ndị ọrụ ibe, Co-SODA oxidized na usoro abụọ enwere ike idozi: n'ime sekọnd, metal ahụ nọrọ paramagnetic (Co²⁺) ebe sọlfọ K-ọnụ XAS gosiri otu thiolate ghọrọ sulfinate; gafere awa, ike ndọta na Co K-eti XAS gosiri igwe na-aga Co³⁺. Nke abụọ thiolate emeghị oxidize n'ihu na ọ dịghị catalytically na-arụ ọrụ mgbagwoju kpụrụ - ekenyere na enweghị axial thiolate.. Nke ahụ bụ peptide dị iche na usoro oxidation dị iche, na ekwesighi ijikọ ya na nsonaazụ Ac-VCTLCSCY; uru ya ebe a bụ ihe atụ maka ihe metallopeptide nwere ike na enweghị ike idozi.
Akwụkwọ nyocha na-enye oke ibu. Nyocha Shalev nke metal-peptide NMR na-ekwu na peptides na-agbanwe nke ọma iji kwekọọ na geometries na-ezighi ezi n'oge nhazi nhazi, na-ebute nsonaazụ nhazi ezighi ezi, na ụdị ndị sitere na peptide nwere ike jikọta ya na njikọ dị ala n'ihi na ọ bụghị ihe niile ejikọtara ọnụ ka echekwabara.. Nke ahụ abụghị arụmụka megide ụzọ; ọ bụ ya mere a ga-eji gụkọta ụdị mgbakọ na mwepụ ọtụtụ spectroscopy ọnụ. Ụdị nke na-ezighị ezi na nkọwapụta, ụzọ a na-enyo enyo na-agwa gị karịa holoenzyme nke ziri ezi n'ihi ihe kpatara na ọ dịghị onye nwere ike ikewapụ ya.
Ihe Cobalt mere n'ezie: Nhazi na Spin States
N'okpuru ọnọdụ anaerobic, UV-visible spectrum nke Co(II)-peptide complex gosipụtara oke mbufe ligand-to-metal chaja na 305 nm (e = 2800 M⁻¹cm⁻¹) na 355 nm (e = 2180 M⁻¹cm⁻¹), Enyere ya na cysteine thiolate-to-Co(II) nnyefe ego. N'ihi na ọmụmụ metallopeptide atụnyere na-etinye thiolate-Co(II) Mmekọrịta na ihe dịka 900-1200 M⁻¹cm⁻¹, ihe ndị a na-ahụ anya na-adaba na thiolates abụọ jikọtara ọnụ, na-ahapụ cysteine nke atọ na-adịghị ike ma ọ bụ na-enweghị nhazi. Njikọ ligand-field adịghị ike na ihe dị ka 615, 685 na 735 nm (ε ≈ 300–400 M⁻¹cm⁻¹) dị elu karịa octahedral Co(II) na-enyekarị, na-atụ aka na ọnụ ọgụgụ nhazi nke anọ ma ọ bụ ise.
A na-etinye apo-peptide na Co(II) mgbe ị na-enyocha nnabata na ~ 685 nm dabara ihe dịka otu saịtị njikọ kwa peptide, na ihe pụtara ìhè Kd,ngwa nke 0.45 µM. Ndị ode akwụkwọ kwuru hoo haa ihe ọnụọgụ ahụ nwere ike iburu: n'ihi na ntinye uche peptide karịrị uru dabara adaba, titration na-emesi ike, ihe dị ka njikọ stoichiometric mana ọ naghị ekpebisi ike nkewa nkewa nke sub-micromolar mgbe niile.. Ọnụ ahịa a tụnyere nke a kọrọ maka Co-NiSOD na Co-SODA peptide complexes.
X-band EPR na 12 K kpebiri akara abụọ. Nke mbụ bụ ntakịrị rhombic low-spin Co(II) atụmatụ na g-ụkpụrụ nke 2.30, 2.25 na 2.03; L-band EPR na 1.3 GHz daa g-nsogbu zuru ezu iji dozie I = 7/2 Ọdịdị hyperfine nke ọma karịa nke ọma. Nke abụọ bụ mgbama mgbaama dị elu dị ala n'etiti 1,000 na 2,000 G nwere isi ntụgharị agbanweela na g' = 5.7 na 4.8, kwekọrọ na otu S = 3/2 usoro. Mmekọrịta nyere ihe dị ka nha nha nha ike maka ha abụọ n'okpuru ọnọdụ anaerobic.
DFT kwadoro geometry tetrahedral N2S2 gbagọrọ agbagọ maka ụdị agba dị elu yana nhazi ise., jiometrị square-pyramidal agbagọ agbagọ maka ụdị ndị dị obere, nwere ụdị isii emepụtara n'ofe atọ enwere ike nke CysSH na-anọpụ iche (Cys2, Cys5 ma ọ bụ 7). Gbakọọ g-ụkpụrụ maka ụdị nhazi ise (1.98-2.57) daa na nkwekọrịta ezi uche dị na ya na oke nnwale 2.03-2.40, na ihe nlereanya ya na thiol na-anọpụ iche na Cys7 megharịrị àgwà axial nke nnwale ahụ g-tensor karịsịa nke ọma. Nkọwa nke ndị ode akwụkwọ - ọtụtụ geometries nhazi nhazi dị n'ime ngbanwe na-agbanwe agbanwe - bụ nkọwa nke mkpokọta na spectroscopy jikọtara ọnụ., ọ bụghị usoro tụrụ atụ.
Oxidation: Ihe gbanwere, Ihe E Mere, na Ihe E Ekpebeghi
Ngosipụta na dioxygen kpochapụrụ Co(II) EPR na-egosi ngwa ngwa, na-enweghị mgbanwe oxygen na-adabere na oke nke elu- na ụdị obere ogho a na-enyocha tupu akara ngosi apụọ. Ntụnyere na Co(II) na nchekwa guzosie ike ihe abụọ: mgbama mgbama dị elu sitere na cobalt aqueous dị iche na mgbama dị na mgbagwoju anya, na ikpughe ikuku anapụtaghị naanị cobalt na peptide. Ọnwụ nke mgbaàmà kwekọrọ na oxidation na EPR-silent low-spin Co(III), na ụdịdị dị iche iche a na-ahụ anya gbanwere UV - ntụgharị d-d n'etiti 600 na 750 nm belatara wee pụọ n'anya ebe ndị egwu ọhụrụ pụtara na ihe dịka 350, 465 na 620 nm, njirimara nke thiolate-to-Co(III) nnyefe ego.
N'akụkụ peptide, q-TOF LC-MS gosipụtara akara kwekọrọ na ntinye nke ihe ruru atọ oxygen atọ mgbe ekpughere ikuku.. Ụdị oxygenated mụbara na ikwu ike na 10 na 15 h, na elu-m/z ọnụ ọgụgụ kacha elu nwere ike dabara na ntinye mmiri. Mgbanwe ndị a dabara na cysteine oxidation na usoro sulfonic na sulfinic acid., Otu mgbanwe ahụ ahụrụ na saịtị ndị na-arụsi ọrụ ike.
Ihe abụọ dị na data anaghị eguzobe, na akwụkwọ akụkọ na-ekwu otú ahụ. Mass spectrometry anaghị ewepụta ọnọdụ oxidation nke etiti igwe. Na usoro nke metal na ligand oxidation enweghị ike guzobe site na data dị ugbu a - nsonaazụ na-egosi na usoro abụọ a na-eme n'oge mmeghachi omume na dioxygen., ọ bụghị nke na-aga mbụ.
Njikwa mkpa dị ka ihe si na ya pụta. Ihe ngwọta nke naanị peptide nọgidere na-enweghị ụcha na mkpughe ikuku mana n'ikpeazụ ọ malitere, enwere ike site na nhazi disulfide. Obere ụdị ikuku oxygen pụtara na-enweghị cobalt, kwekọrọ na oxidation cysteine dị ala n'oge njikwa sample. Nke ahụ bụ ihe akaebe nke ọmụmụ ihe na-atụ anya na ndabere oxidation ndabere ka a na-atụ anya ka ọ bụrụ echiche efu.
Ndị ode akwụkwọ na-ebulitekwa ịkpachara anya n'ihu: Ogologo ikuku oxygen na-adịte aka nwere ike ịmepụta ụdị oxidized karịa, nwere ike ịgụnye gburugburu bis-sulfinic-acid nke yiri oke oxidized, NHase adịghị arụ ọrụ. Maka onye ọ bụla na-agbatị oge incubation, nke ahụ bụ amụma iji nwalee, ọ bụghị ihe edoziziri.
Nkwenye nyocha: Kedu usoro na-aza ajụjụ
Usoro ọ bụla n'ime ọmụmụ ihe a na-aza otu ajụjụ ma kpuru ìsì Peptides akpọrọ ndị ọzọ. Ịgụ akwụkwọ metallopeptide pụtara ịmara nke bụ.
UV – nnabata a na-ahụ anya na-aza ma thiolate ejikọtara na igwe yana ole ole, site na LMCT band ike megide ~900-1200 M⁻¹cm⁻¹ per-thiolate benchmark. Ọ nweghị ike idozi ọnọdụ spine ma ọ bụ geometry, ma ọ nweghị ike ịgwa gị nke cysteine kechiri.
MCD na EPR na-aza ihe steeti ntụgharị dị na kedu oke; ike EPR agbakwunyere nyere ihe dị ka oke mgbago dị elu/obere nhata, na mkpebi L-band kwadoro ọrụ dị ala site na nhazi hyperfine. Mana EPR kpuru ìsì na EPR-silent Co(III), ya mere mwepu nke mgbaàmà kwekọrọ na oxidation na-enweghị ịbụ nha kpọmkwem ya.
LC-MS na-aza ole oxygen dị na peptide na otú nke ahụ si aga n'ihu na oge. O nweghị ihe na-ekwu banyere nke sọlfọ na-ebu ha, ọ dịghịkwa ihe ọ bụla gbasara ọnọdụ oxidation metal. A na-edekọ oke oke nke oxidative-modification mass spectrometry nke ọma - mgbanwe-kpọmkwem workflows mara na ikenye mgbanwe na ihe fọdụrụ na-achọkarị nkọwa MS/MS, na n'ịkọwapụta oxidized na ụdị unoxidized sitere na chromatogram ion ewepụtara na-ewere ionization na arụmọrụ nnyefe yiri ya..
DFT na-aza ụdị nhazi nke geometrically na ike nwere ike ịpụta, ma naanị n'ihi na truncated ụdị n'ezie gbakọọ - ọ bụghị maka zuru peptide na ngwọta.
N'akụkụ njikọ, nlele ntọhapụ nke ọmụmụ ihe na-enye nkuzi: free thiols na 0.76 MM apo-peptide ngwaahịa kwadoro site na nyocha Ellman, na absorptivity nke molar na 280 nm (1,096 M⁻¹cm⁻¹) emebere ya site n'usoro ọkọlọtọ ka e wee mara itinye uche karịa ka e were ya. Ọ bụghịkwa usoro njirimara metalloprotein, ma ha abụọ bụ ihe achọrọ maka otu.
Iwu bara uru: ọ dịghị otu usoro na ndepụta a zuru ezu. Akụkọ njirimara nke na-enye naanị otu emebeghị ka metallopeptide.
Ịdị ọcha, njirimara, Ọdịnaya na Oxidation State bụ ajụjụ anọ dị iche iche
A sample nwere ike ịbụ 98% dị ọcha site na HPLC ma ka bụrụ nke a na-apụghị iji ya mee ihe maka Co(II) nnwale. Palmitoyl Pentapeptide-4 Paradox ahụ pụtara ìhè na-agbaze ozugbo ajụjụ anọ ahụ kewapụrụ.
Ịdị ọcha bụ mpaghara pasentị site na HPLC. Ọ na-ekwu ole n'ime ihe ndị na-anabata UV na-apụta dị ka ọnụ ọgụgụ kachasị elu. O kwughị ihe ọnụ ọgụgụ ahụ bụ, ọ dịghịkwa ọdịiche dị n'etiti cysteine nke na-ebelata site na nke emelarị oxidized ma ọ bụrụ na ha abụọ elu ọnụ.
njirimara bụ nkwenye nhazi orthogonal. Ebe a pụtara uka spectrometry, na LC-MS nke ọmụmụ ihe na-egosi ihe kpatara otu nha oke nwere oke: ozugbo ikuku oxygen na-ebufe oke site na 16 Na kwa oxygen, peptide oxidized na peptide dị iche nwere ike ịgbagwoju anya ma ọ bụrụ na mkpebi oke na ọnọdụ chromatographic zuru oke..
Ọdịnaya, ma ọ bụ peptide net, bụ uka itule - peptide plus counterion, mmiri na nnu. Ọ dị mkpa ebe a n'ihi na stoichiometry nke na-ejikọta titration na-adabere na ịmara ole peptide dị n'ezie na cuvette.. Nleta aha aha agbakọrọ site na ntụ ntụ atụnyere na-enweghị mkpebi ọdịnaya nwere ike ịgbanyụ ya nke ọma iji gbanwee usoro njikọ dabara adaba..
Oxidation steeti bụ ihe onwunwe nke cysteine na ígwè nke na-adịghị HPLC ma ọ bụ emebibeghị ọnụ ọgụgụ na-akọ nke ọma.. A lyophilized ntụ ntụ bụ 98% dị ọcha site na HPLC nwere ike ịnwe akụkụ dị nta nke cysteines nke oxidized na ụdị sulfonic ma ọ bụ disulfide site na njikwa na lyophilization.. Nke ahụ ga-egosi dị ka oke dịtụ elu, oge njigide agbanwere, ma ọ bụ naanị titer free-thiol site na Ellman's. Nleba anya nke ọmụmụ nke obere ụdị oxygenated na enweghị cobalt, ekwuru na njikwa sample, bụ ihe na-egosi na nzụlite a dị adị.
Usoro mkpebi na-esote: maka ọrụ metallopeptide nke nwere mmetụta oxidation, ezipụta ọdịnaya thiol efu yana profaịlụ oxidation dị ka njirisi ntọhapụ, ọ bụghị naanị HPLC ịdị ọcha na nlele uka. Ụkpụrụ nyocha dị n'azụ nkọwapụta ndị dị otú ahụ bụ otu ihe ahụ e depụtara na ya I Q2(R2), nke na-emeso njirimara, ịdị ọcha na nyocha dị ka njirimara arụmọrụ dị iche iche karịa ndị na-agbanwe agbanwe.
Ijikwa ígwè na Oxygen-Sensitive Workflow
Ihe ọmụmụ ọmụmụ a kapịrị ọnụ na kwesịrị iṅomi kpọmkwem. A kwadebere ihe nlele peptide niile n'okpuru ọnọdụ anaerobic n'ime igbe mkpuchi ihu dị nro nke Coy. A gbazere peptide na degassed 10 mM N-ethyl morpholine (Ọ BỤGHỊ) buffer na pH 8.0, na-enye enweghị agba 0.76 MM apo-peptide ngwaahịa nke soluble wee kwado ya maka thiols efu site na nyocha Ellman.. Otu nhata nke CoCl₂ · 6H₂O, agbazere n'otu nchekwa n'otu pH, agbakwunyere na peptide buffered.
Ihe na-esote kemịkalụ nhazi karia site na mpempe akwụkwọ kwesịrị ikwupụta dị ka echiche imewe. Usoro nke mgbakwunye na metal-to-peptide ratio na-ekpebi ma e bu n'obi 1:1 ụdị mgbagwoju anya ma ọ bụ ngwakọta na-eme; ya mere a na-agba titration karịa ka e were ya. Nhọrọ nchekwa dị mkpa n'ihi na nhazi ihe nchekwa na-asọ mpi maka igwe, na NEM ahọpụtara dị ka ihe nchekwa morpholine na-enweghị nhazi. Na peptide-naanị mmiri ozuzo na ikpughe ikuku pụtara nguzobe disulfide bụ ụzọ asọmpi na-ewepụ peptide na ngwọta tupu ya enwee ike jikọta cobalt - ọwa ọnwụ nke na-eji oge eme ihe.. Pancreatic polypeptide
Oghere nke eziokwu: akwụkwọ akụkọ anaghị ekwupụta njikarịcha n'usoro nke nha igwe, usoro mgbakwunye ma ọ bụ njirimara nchekwa. Onye na-agụ akwụkwọ na-amụba nke a na-agbatị usoro ahụ, ọ bụghị iṅomi ya, na kwesịrị na-emeso nke ọ bụla n'ime ndị mgbanwe dị ka ihe na-akwado na ha usoro kama a chọrọ eluigwe na ala.
Kedu ihe R&D Lead Kwesịrị Ịjụ Tupu Ịnye Metallopeptide Oxidation-Sensitive Metallopeptide
Ajụjụ ndị dị n'okpuru bụ ndị na-ekpebi ma metallopeptide zụrụ na-akwado nnwale ahụ. A na-aza nke ọ bụla na mkparịta ụka nkọwapụta.
Usoro na ụdị. A na-edekọ usoro nke ọma - gụnyere N-terminal acetyl na ihe ọ bụla na-enweghị isi - edekọtara dị ka ndekọ ejiri ụdị., na asambodo na-ekwu ya? Ndezigharị usoro na-enweghị ụzọ ụdị a na-apụghị ịgbanwe agbanwe bụ ihe na-ebutekarị nnwale metallopeptide enweghị ike imepụtagharị..
Ọnọdụ Cysteine . Gịnị bụ free-thiol ọdịnaya na mwepụta, kedu usoro, na ụdị oxidation nke akọwapụtara dị ka oke? Nke a bụ ajụjụ nyocha nke njikwa-oxidation nke ọmụmụ ihe na-eme ka enweghị mkparịta ụka. Acetyl Hexapeptide-1 Onye nrụpụta
njirimara. Enwere nkwenye orthogonal gafere otu nha oke, na-eme ka uka spectrometry na-edozi oghere oxygen? Usoro na-enweghị ike ikewa +16 Site na nne na nna enweghị ike ịkọ profaịlụ oxidation.
Ọdịnaya. A na-akọ ọdịnaya peptide net dị iche na ịdị ọcha HPLC, na bụ counterion form kwuru? Stoichiometry na titration ejikọta na-adabere na nke mbụ; omume nchekwa na asọmpi igwe nwere ike ịdabere na nke abụọ.
Ijikwa. A na-enye ihe a n'ụdị na nkwakọ ngwaahịa na-akwado nhazigharị anaerobic, na akwụkwọ ahụ na-ekwu otú ahụ? Otu ntụ ntụ lyophilized dị mma, ma nzọụkwụ nrụzigharị bụ ebe ikuku oxygen na-abanye.
Ngwungwu nyocha. A na-enye chromatogram raw na ụdịdị dị iche iche nwere ihe nchọpụta usoro - kọlụm, gradient, ogologo ife efe, oke oke - kama tebụl nchịkọta? Enweghị ike inyocha tebụl nchịkọta ọzọ mgbe nsonaazụ dị njọ.
Ọnụ ọgụgụ. Ọ bụrụ na ihe nlereanya ahụ gụchara site na milligrams na nnukwu batches, enwere ihe ndabere maka ịtụ anya profaịlụ oxidation ga-ejide? Usoro oxidation-sensitive bụ kpọmkwem ebe mgbanwe usoro na-egosi na mbụ.
Onye na-ebubata ihe na-enweghị ike ịza ajụjụ cysteine-ọnọdụ na-agwa gị ihe gbasara ikike nyocha dị n'azụ asambodo ahụ.. Nke ahụ bụ azịza kacha enye nkọwa na ndepụta ahụ.
Ebe Ihe Nlereanya Akwụsị
Ihe peptide abụghị enzyme. Ọ nweghị β-subunit, protein activator, ngalaba nhazi nke abụọ na ụzọ subunit-swapping maturation ụzọ. Ntụnyere ihe ọmụmụ a na usoro nke ala bụ site na ntule nke spin state na ligand modification, ọ bụghị site na ọrụ catalytic - ọ dịghị hydration nitrile ka a tụrụ ebe a.
Usoro nke metal megide ligand oxidation ka edozibeghị na sistemụ a, dị ka ndị odee si kwuo. Njide mgbe niile bụ uru pụtara ìhè a tụrụ n'okpuru ọnọdụ na-anabataghị mkpebi siri ike nke obere micromolar.. Ụdị DFT a na-agbajikwa ma na-edozi ya na-aga n'ihu. Na kemịkalụ oxidation ahụrụ bụ ihe na-eme ebumnuche dịpụrụ adịpụ na nchekwa - nkwupụta gbasara mmeghachi omume nke motif., Ọ bụghị nkwupụta gbasara usoro maturation intracellular.
Ọ dịghị nke ọ bụla n'ime ihe ndị a na-ebelata nsonaazụ ya. Ọ na-akọwa ihe nsonaazụ na-akwado: na usoro echekwara naanị na-ebu ozi kemịkalụ zuru oke iji bind Co(II) na ijikọta ọla kọpa na cysteine oxidation na ikpughe dioxygen.
Mmechi
Ihe peptide fọdụrụnụ asatọ dị mkpa n'ihi na ọ na-atụgharị ajụjụ ntozu okè na-enweghị ike ịbanye n'ime usoro mgbanwe mgbanwe - nọmba nhazi., ogho steeti, ọnụọgụ oxygen, ọnọdụ free-thiol - nke ọ bụla nwere usoro na-akọ na ya na ntụpọ kpuru akwụkwọ. Nchoputa na conserved motif alone binds Co(II) ma na-enweta ọla kọpa na cysteine oxidation na mkpughe ikuku bụ nkwupụta gbasara ozi kemịkalụ nke usoro ahụ..
Ọ bụ naanị dị ka ntụkwasị obi dị ka iguzosi ike n'ezi ihe nke usoro ihe atụ, steeti oxidation na akwụkwọ nyocha. Maka ndị otu na-ewu ụdị ihe nlereanya a, nzọụkwụ ọzọ bụ mkparịta ụka nkọwapụta kama ịzụrụ ihe: kọwaa ọdịnaya thiol efu, oke oxidation, net peptide net na ngwugwu data raw tupu emee milligram mbụ. MOL mgbanwe na-enye peptides omenala na akwụkwọ HPLC na MS, Mkpebi ọdịnaya peptide nhọrọ yana nyocha mgbochi, na njikwa anaerobic dakọtara na arịrịọ; ikike ndị ahụ dabara na ajụjụ nkọwapụta dị n'elu, na ndị na-agụ akwụkwọ kwesịrị ịtụle mmasị azụmahịa ahụ mgbe ha na-enyocha ntụzịaka nhọrọ ndị na-ebubata ngwaahịa.
MOL Mgbanwe bụ sayensị peptide R&D nhazi ma na-ebunye peptides omenala na ọrụ nyocha nyocha. Ndị na-agụ akwụkwọ kwesịrị ịtụle mmasị azụmahịa a mgbe ha na-enyocha ntụzịaka nhọrọ nke ndị na-ebubata ihe n'isiokwu a.
Isi mmalite: Ọmụmụ cobalt metallopeptide izizi.
