Uphononongo lojongano lwe-intersubunit yetshaneli yeproton evulekileyo ye-Hv1 kunye neprobe edityaniswe ngokwe-allosterically

Enkosi ngokundwendwela i-Nature.com.Inguqulelo yesikhangeli oyisebenzisayo inenkxaso enyiniweyo ye-CSS.Ngowona mava angcono, sicebisa ukuba usebenzise isikhangeli esihlaziyiweyo (okanye ucime imo ehambelanayo kwi-Internet Explorer).Okwangoku, ukuze uqinisekise inkxaso eqhubekayo, siya kubonisa isayithi ngaphandle kwezitayile kunye neJavaScript. Ijelo le-proton ye-voltage ye-Hv1 yi-dimeric complex equkethe i-domain ezimbini ze-voltage-sensing (VSDs), nganye iqulethe i-gated proton permeation pathway.I-Dimerization ilawulwa yi-cytoplasmic coiled-coil domain. imeko evulekileyo kuzo zombini ii-VSDs iyaziwa ngokuba yenzeke ngentsebenziswano; nangona kunjalo, kuncinci okwaziwayo malunga neendlela ezisisiseko.I-intersubunit interfaces idlala indima ebalulekileyo kwiinkqubo ze-allosteric; nangona kunjalo, ujongano olunjalo aluzange luchongwe kwiitshaneli ze-Hv1 ezivulekileyo. Apha sibonisa ukuba i-2-guanidinothiazole i-derivatives ivimba ii-VSD ezimbini ze-Hv1 ngendlela yokubambisana kwaye isebenzise enye yale ngqungquthela njenge-probe yokudibanisa i-allosteric phakathi kwee-subunits ezivulekileyo.Sifumene ukuba i-extracellular isiphelo seqhekeza lokuqala le-transmembrane ye-VSD senza ujongano lwe-intersubunit olulamla ukudityaniswa phakathi kweziza ezibophelelayo, kanti i-coiled-coil domain ayibandakanyekanga ngokuthe ngqo kule nkqubo.Siphinde safumana ubungqina obuqinileyo bokuba i-channel-selective filter control control blocker-binding cooperativity. . Iziteshi zeproton ezine-voltage-gated zidlala indima ebalulekileyo kwizinto ezahlukeneyo eziphilayo, ukusuka kwi-phytoplankton ukuya ebantwini1.Kwiseli ezininzi, ezi tshaneli zilamla i-proton efflux ukusuka kwi-membrane ye-proton kwaye zilawula umsebenzi we-NADPH oxidase.Ukuphela kwejelo leproton eyaziwayo yombane-gated ebantwini. yi-Hv1, eyimveliso ye-HVCN1 gene2,3.Hv1 (aka VSOP) iboniswe ukuba idlale indima kwi-B cell proliferation4, ukuveliswa kweentlobo ze-oksijini ezisebenzayo yi-innate immune system5,6,7,8, i-sperm cell. motility9 kunye nokulawulwa kwe-pH yolwelo lomphezulu womoya10. Eli tshaneli libandakanyeka kwiintlobo ezininzi ze-Overexpressed kwiintlobo zomhlaza ezifana ne-B-cell malignancies4,11 kunye ne-breast and colorectal cancers12,13.Umsebenzi ogqithisileyo we-Hv1 ufunyenwe ukwandisa amandla e-metastatic yeeseli zomhlaza 11, 12.Engqondweni, i-Hv1 ibonakaliswe nge-microglia, kwaye umsebenzi wayo ubonakaliswe ukwandisa umonakalo wobuchopho kwiimodeli ze-stroke ye-ischemic. Iprotheyini ye-Hv1 iqulethe i-domain ye-voltage-sensing domain (VSD), equkethe amacandelo amane e-transmembrane ebizwa ngokuba yi-S1 ukuya kwi-S414. I-VSD ifana neendawo ezihambelanayo ze-voltage-gated Na +, K + kunye neCa2 + iziteshi kunye ne-voltage-sensitive phosphatases, njenge-CiVSP evela. I-Ciona gutis15.Kulezi ezinye iiprotheni, i-C-terminus ye-S4 ifakwe kwimodyuli yesiphumo, i-pore domain okanye i-enzyme.Kwi-Hv1, i-S4 idibaniswe ne-coiled-coil domain (CCD) ebekwe kwicala le-cytoplasmic ye-membrane. .Itshaneli yi-dimeric complex equkethe ii-VSD ezimbini, nganye iqulethe i-gated proton permeation pathway16,17,18. Ezi zincinci zimbini ze-Hv1 zifunyenwe zivuleke ngokubambisana19,20,21,22, zicebisa ukuba ukudibanisa i-allosteric kunye ne-inter-subunit interactions play indima ebalulekileyo kwinkqubo yokungena.I-interface phakathi kwe-subunits ngaphakathi kwe-coil coil domain ichazwe kakuhle ngenxa yokuba izakhiwo zekristal zeendawo ezimbini ezizimeleyo zifumaneka22,23.Ngakolunye uhlangothi, i-interface phakathi kwe-VSDs ngaphakathi kwe-membrane ayikho. iqondwa kakuhle.Isakhiwo sekristale yeprotein ye-chimeric ye-Hv1-CiVSP ayiboneleli ngolwazi malunga nolu jongano, njengoko intlangano ye-trimeric ye-crystallized channel complex ingabangela ukutshintshwa kwe-CCD ye-Hv1 yomthonyama yi-yeast leucine zipper GCN424. Uphononongo olutshanje lwe-subunit yentlangano ye-Hv1 channel yagqiba ukuba ezimbini ze-S4 helices zitshintshela kwi-CCD ngaphandle kokuphazamiseka okukhulu kwesakhiwo sesibini, okubangelwa yi-helices emide eqala kwi-membrane kunye neprojekthi kwi-cytoplasm. olu phononongo lucebisa ukuba ii-VSD ze-Hv1 ziqhagamshelane ecaleni kwecandelo le-S4. Nangona kunjalo, ezinye izifundo zicebise ujongano olulolunye phakathi kwe-VSDs.Olu nxibelelwano lubandakanya amacandelo e-S1 17, 21, 26 kunye neziphelo zangaphandle ze-S2 segment 21.Isizathu esinokubakho sokuphikisana Iziphumo zezi zifundo kukuba ukudibanisa i-allosteric phakathi kwe-VSDs kwavavanywa ngokunxulumene nenkqubo yokungena, exhomekeke kwiindawo ezivaliweyo nezivulekileyo, kunye nojongano phakathi kwe-VSDs lunokwahluka kwiinguqu ezahlukeneyo zemo kwi-conformation. Apha, sifumene ukuba i-2-guanidinothiazole inqanda iziteshi ze-Hv1 ngokuzibophezela ngokubambisana kwii-VSD ezimbini ezivulekileyo, kwaye isebenzise enye yeekhompawundi, i-2-guanidinobenzothiazole (GBTA), ukuhlolisisa intsebenziswano phakathi kwama-subunits kwindawo evulekileyo. i-interface.Sifumene ukuba i-GBTA yokubopha i-curve inokuchazwa kakuhle yimodeli yobungakanani apho ukubophelela kwe-inhibitor kwi-subunit enye kubangela ukwanda kobudlelwane obubophelelayo be-subunit ekufutshane.Siphinde safumanisa ukuba iintsalela ze-D112, izihlungi zokukhetha itshaneli 27, 28 kunye nenxalenye guanidine derivative indawo yokubopha 29 ulawulo GBTA ebophelelayo cooperativity.Sibonisa ukuba ukubophelela intsebenziswano igcinwe kwi Hv1 dimer, apho iCCD isahlula icandelo S4, ecebisa ukuba intersubunit ujongano kwiCCD ayenzi ngokuthe ngqo. I-mediate allosteric coupling phakathi kwe-GBTA-binding sites.Ngokwahlukileyo, sifumanisa ukuba i-S1 fragment iyingxenye ye-interface phakathi kwe-subunits kwaye iphakamisa ilungiselelo le-VSDs ekufutshane kunye nokuphela kwe-extracellular ye-S4 helix kude nombindi we-dimer ukuvumela. iqhekeza le-S1 libe kwindawo evulekileyo. I-molekyuli encinci inhibitors ye-Hv1 iluncedo njengamachiza e-anticancer kunye ne-neuroprotective agents.Nangona kunjalo, ukuza kuthi ga ngoku, iikhompawundi ezimbalwa ziye zakwazi ukunqanda i-channel30,31,32,33.Phakathi kwabo, i-2-guanidinobenzimidazole (2GBI, i-compound [1] kwiFig. I-1a) kunye ne-derivatives yayo ifunyenwe ukuvimba i-VSD29,32 i-permeation ye-proton ngokusebenzisa i-channel.Ukubotshwa kwee-compounds ezinjalo kucatshangelwa ukuba kwenzeke ngokuzimeleyo kwii-subunits ezimbini ezivulekileyo. ngaphambili kubonisiwe ukuba inhibit Hv1 phantse ngempumelelo njenge 2GBI xa ivavanywa kuxinaniso 200 μM (Figure 1b) .Sivavanye ezinye derivatives thiazole kwaye safumanisa ukuba ezinye zazo inhibite ijelo kunye potency efanayo okanye ngaphezu GBTA (Fig. 1 kunye Supplementary). umbhalo) .Sinqume i-concentration curves ye-concentration curves of four thiazole derivatives (GBTA kunye ne-compounds [3], [6] kunye ne- [11], i-Fig. 1c) kwaye yafumanisa ukuba i-steeper kunezo ze-2GBI.I-Hill coefficients (h) of thiazole derivatives ukusuka 1.109 ± 0.040 ukuba 1.306 ± 0.033 (Fig. 1c kunye Supplementary Fig. 1) Ngokwahlukileyo, Hill coefficient for 2GBI kwaba 0.975 ± 0.024 29, Fig. 2a kunye Fig. 1). I-Hill coefficient ngasentla kwe-1 ibonisa intsebenziswano ebophelelayo.Kuba iyunithi nganye ye-Hv1 ine-inhibitor yayo. indawo ebophelelayo,29,32 siye saqiqa ngelithi ukudityaniswa kwe-thiazole ephuma kwiyunithi enye kunokuphucula ukudityaniswa kwemolekyuli yesithinteli sesibini kwi-subunit ekufutshane.I-GBTA yayiyikhompawundi yovavanyo ene-Hill coefficient.Ngoko ke, sikhethe le khompawundi ukuba funda ngakumbi indlela yokubopha i-synergy kwaye yasebenzisa i-2GBI njengolawulo olubi lwereferensi. (a) Iikhompawundi zovavanyo: [1] Reference Hv1 inhibitor 2-guanidino-benzimidazole (2GBI).[2] I-2-guanidino-benzothiazole (GBTA), [3] (5-trifluoromethyl-1,3-benzothiazol-2-yl)guanidine, [4] naphtho[1,2-d][1, 3] Thiazol-2-yl -guanidine, [5] (4-methyl-1,3-thiazol-2-yl) guanidine, [6] (5-bromo-4-methyl-1,3-thiazol- 2-yl) guanidine, [7] famotidine, [8] 2-guanidino-5-methyl-1,3-thiazole-4-carboxylic acid ethyl ester, [9] 2-guanidino-4-methyl Ethyl-1,3-thiazole-5-carboxylate, [10] ](2-guanidino-4-methyl-1,3-thiazol-5-yl)ethyl acetate, [11]1-[4-(4 -Chlorophenyl)-1,3-thiazol-2-yl]guanidine, [ 12]1-[4-(3,4-dimethoxyphenyl)-1,3-thiazol-2-yl]guanidine .(b) Ukuvinjelwa komsebenzi we-Hv1 yabantu nge-guanidinothiazoles ebonisiweyo kunye ne-compound ye-reference 2GBI (imivalo eluhlaza okwesibhakabhaka) .Imisinga yeproton ye-Hv1 yalinganiswa kwiiplagi zangaphakathi-ngaphandle ze-Xenopus oocytes ekuphenduleni ukuchithwa kwepolarization ukusuka kwikhono lokubamba le--80 mV ukuya kwi-+120 mV.I-inhibitor nganye yongezwa ebhafini kugxininiso lwe-200 μM.pHi = pHo = 6.0 .Iidatha zithetha ±SEM (n≥4) .(c) Ukunyanzeliswa okuxhomekeke kwi-concentration ye-Hv1 yabantu nge-compounds [2], [3], [6] kunye ne- [11].Inqaku ngalinye limele inhibition ephakathi ± SD ye-3 ukuya kwimilinganiselo ye-15. Umgca yi-Hill fit esetyenziselwa ukufumana amaxabiso abonakalayo e-Kd axelwe kwiTheyibhile eyoNgezelelweyo ye-1. I-coefficients ye-Hill yamiselwa ukusuka kwimilinganiso echazwe kwi-Supplementary Fig. 1: h (1) = 0.975 ± 0.024 h (2) = 1.306 ± ± 1.306 ± 0.033, h (3) = 1.25 ± 0.07, h (6) = 1.109 ± 0.040, h (11) = 1.179 ± 0.036 (jonga IiNdlela). (a,b) I-Compounds 2GBI kunye ne-GBTA inhibited dimeric kunye ne-monomeric Hv1 ngendlela exhomekeke kugxininiso.Inqaku ngalinye limele inhibition ephakathi ± SD ye-3 ukuya kwi-8 yemilinganiselo kunye ne-curve yi-Hill fit.I-Hill coefficients (h) eboniswe i-histograms ye-inset yamiselwa ukusuka kwimilinganiso exelwe kwiiFig ezoNgezelelweyo 3 kunye ne-4.Impendulo yoxinaniso ye-GBTA eboniswe kwi-(a) iyafana naleyo ikuFig. ukudityaniswa ngokubambisana kwe-GBTA ukuya kwi-dimeric Hv1.Umgca omnyama oqinileyo umele ukufaneleka kwidatha yovavanyo nge-equation (6), echaza imodeli ebophelelayo eboniswe kwi-(d). -i-dissociation equilibrium curves yeziganeko ezibophezelayo zokuqala nezesibini, ngokulandelanayo (I-Sub 1: OO + B ⇄ BO *, Kd1 = 290 ± 70 μM; I-Sub 2: BO * + B ⇄ B * O *, Kd2 = 29.3 ± 2.5 μM .(d) Umzobo weSchematic wendlela ecetywayo yebhloko ye-Hv1. Kwimeko ye-GBTA, ukubophelela kwi-subunit enye evulekileyo kwandisa ukuhambelana kwe-subunit evulekileyo ekufutshane (Kd2 0.05) kwi-Hill coefficient ye-GBTA ebophelela kumajelo e-GGG xa kuthelekiswa nohlobo lwasendle (Umfanekiso 5c), ebonisa ukuba nangona indima yayo ekugcineni amasuntswana amabini kunye abalulekile, kodwa iCCD ayilamleli ngokuthe ngqo udibaniso lweallosteric phakathi kweendawo ezibophelelayo ze-GBTA. (a) Umzobo weSchematic we-Hv1 dimer enokuguqulwa kwe-triglycine kwisiphelo sangaphakathi se-S4 eyenzelwe ukuphazamisa udibaniso lwe-intersubunit elamlwa yi-cytoplasmic coiled-coil domain (iintolo eziblue). ukuguqulwa okubonakalisiweyo, okwenzelwe ukuvavanya iziqwenga ze-S1 ezibandakanyekayo ekudibaniseni phakathi kwee-subunits (iintolo eziluhlaza).(c-h) I-2GBI (cyan) kunye ne-GBTA (ebomvu ebomvu) inqanda ukwakhiwa okubonisiweyo ngendlela exhomekeke kugxininiso.Inqaku ngalinye limele inhibition ephakathi. ± SD ye-3 ukuya kwi-10 imilinganiselo. Ijika yayiyi-Hill fit esetyenziselwa ukufumana amaxabiso acacileyo e-Kd (bona iTheyibhile eyoNgezelelweyo 1). I-Hill coefficients kwi-interpolated histograms yamiselwa njengoko kuchaziwe kwicandelo le-Methods (jonga i-Supplementary Figs 3 kunye ne-4) . I-Hv1 WT iboniswa njengemigca edayiweyo.Iiinkwenkwezi zibonisa umahluko obalulekileyo ngokwezibalo phakathi kweendlela eziguqukayo kunye ne-WT (p 0.05, Fig. 5d, i). ). Ngakolunye uhlangothi, ukuguqulwa kwe-D123A kunciphise kakhulu i-Coefficient ye-Hill ye-GBTA (p 0.05/14). Ukusukela ekunciphiseni umrhumo kwindawo ye-123 kuzo zombini ii-subunits kubangele utshintsho oluqinileyo kwi-GBTA ebophelelayo intsebenziswano, ngelixa ukubuyisela umva intlawulo kuwo omabini amacandelo anesiphumo esincinci kuphela, sandise uhlalutyo ukuba lubandakanye iyunithi enye kuphela kunye nomjelo wokuguqula intlawulo. yenziwe i-Hv1-dimers edibeneyo kunye ne-D123R endaweni ye-C-terminal subunit (Fig. 5b) kwaye yalinganisa i-concentration-response inhibition yi-GBTA kunye ne-2GBI. ye-wild-type Hv1 (p 0.05). Siphinde safumanisa ukuba ngokungabikho kwe-βME, i-Hill coefficient ye-GBTA ebophelelayo kwi-D112E I127C Hv1 dimer yayiphezulu kakhulu kunokuba ilinganiswe phambi kokunciphisa ama-agent (Umfanekiso 6e kunye ne-Supplementary Fig. 4), ebonisa ukuba i-D112E yokuguqulwa ayizange iphelise ukwanda kwe-GBTA ebophelelayo intsebenziswano ngenxa yokudityaniswa kwe-cysteine ​​​​127.I-Coefficient ye-Hill ye-2GBI ebophelelayo kwi-D112E, i-I127C Hv1 dimers nayo ayizange ichaphazeleke kakhulu kukuguqulwa kwe-D112E (Umfanekiso 1) .6d kunye ne-Supplementary. Umzobo 3). Zithathiwe kunye, ezi ziphumo zibonisa ukuba ukubopha kwe-GBTA kuphuculwe lunxibelelwano phakathi kweziphelo zangaphandle zamaqhekeza e-S1 kwiiyunithi ezikufutshane ze-Hv1 ngokusebenzisana okunomtsalane kwe-electrostatic okanye ngokwenziwa kweebhondi ezidityanisiweyo phakathi kwe-cysteines endaweni yokudityaniswa kwe-allosteric phakathi kweesayithi zonyuka kunye neziphumo zokubophelela intsebenziswano. Ngelixa impembelelo yokunxibelelana kwe-electrostatic ekhangayo kwintsebenziswano inokupheliswa ngokuguqula i-D112E, isiphumo se-covalent bonds ayikwazi. Ekuhloleni kwethu indawo yeekhemikhali ekhoyo yokubopha i-guanidine derivatives kwi-Hv1 channels, sifumene ukuba i-2-guanidinothiazoles efana ne-GBTA inokuxhomekeka koxinzelelo oluphezulu kune-2-guanidinobenzimidazoles (Umfanekiso 1c) .I-Hill coefficient analysis of GBTA ebophelelayo kuzo zombini i-dimeric kunye neziteshi ze-monomeric (umzobo 2a, b) kunye ne-dimeric channel apho i-subunit enye yayiboshwe ngaphambili kwi-inhibitor (Umfanekiso we-4) yasikhokelela ekubeni sigqibe kwelokuba ukuvinjelwa kwe-Hv1 nge-GBTA Isenzo yinkqubo ye-synergistic, kwaye iindawo ezibophelelayo zeekhompawundi kwii-subunits ezimbini zidibene ngokulinganayo.Ukufumanisa ukuba i-GBTA ibophelela kwitshaneli evulekileyo, njengoko kubonisiwe ngaphambili kwi-compound ehambelana ne-2GBI32, iphakamisa ukuba ukudibanisa i-allosteric kunokuhlolwa ngokukodwa kwimeko evulekileyo.Imodeli yethu yokubambisana ngokubambisana wakwazi ukuchaza ubungakanani be-HV1 nge-GBTA (Fig) kwaye uchaze iimpembelelo ezahlukeneyo ze-2GBI kunye ne-GBTA ekuboleni imisele ye-Membrane, exhasa ukutolika kwethu kwenkqubo yokubopha. Elona chibi liphezulu elinezinto ezinokufezekiswa kwiproteni ye-Advatic ezinamaziko amabini okubopha (njenge-HV1) i-GBTA ilingane no-1.31, olwanda kwi-1.88 kwi-HV1 I12C.I Amandla asimahla esimahla, umahluko phakathi kwamandla amahlathi acocekileyo kwaye ophakamileyo (jonga iindlela ze-fitinity / i-1,3 kcal / mole kwimeko ye-HV1 i127c.Ing Ioksijini ukuya kwi-hemoglobin ngowona mzekelo udumileyo nowafunda kakuhle kwinkqubo yokusebenzisana3.form ye-hemoglobin (i-temomer enezindlu ezihamba-nye), i-tetromer enezinto ezinamatyala ukusuka kwi-1.26 ukuya kwi-3.64 I-KCal / Mol, kuxhomekeke kwiimeko zovavanyo3.Ukusasazwa ngamandla e-GBTA ekubopheleleni kwi-HV1 ibophelele kwi-HV1 ibophelelekile kwi-O2 eyahlukileyo kwi-O2 ye-O2 eyahlukileyo kwiinkqubo ezimbini eziqwalaselweyo. Kwimodeli yethu yokuhamba, ukubopha kweemolekyuli ze-GBTA ukuya kuthi ga kwi-Tounit enye kukhokelela ekuboleni kwendawo ebophelelayo yendawo yokubopha okubangelwa yindawo yokuqala yokubopha (i-Infuzisiwe) ikhokelela Utshintsho kunxibelelwano phakathi kophuhliso.in Impendulo kolu tshintsho, ukuphanziswa kweendawo ekufuphi ne-GBTA ukubopha i-GBTA yokubopha i-Atving Afficity.D112 bekade baboniswa ukuba yinxalenye yendlela ye-HV1 proton ye-HV Iziphumo zethu zibonisa ukuba iifilitha zokukhetha i-HV1 ye-HV1 idityaniswa ngokudibeneyo kwi-veral yendawo ye-GBTA kunye nendawo yendawo ye-D112, D123 kwaye i127 kwi-HV2 VSD esekwe Kwisakhiwo sekristali ye-HV1 . Iindawo ezixineneyo ze-GBTA ziboniswa njengee-ovals ezingwevu. Kumgangatho obini wokucwangciswa kwe-dimer obonakalayo njengoko kubonwe kwicala le-membrane inqwelomoya.On Iphaneli yasekhohlo, i-Dimer ye-Demer yenziwa ngu-Hixds kwi-Axis i-Axis ijikeleze i-Axis Indila, ehamba kunye Ukwahlula-hlula iziphelo zangaphandle zee-helilis ezimbini ze-S4 (iintolo ezihlanjisiweyo), kuvelisa ilungiselelo eliboniswe ngasekunene.in lo uqwalaselo, i-DARDECT ivunyelwe ukuba isondele. (c) Kumgangatho we-Dimer ofunyenwe kwi-4G80 ye-crystal ye-Crystal Iziphumo zethu zibonisa ukuba ukusebenzisana kwe-electrostatic ye-elektrostic kwi-123 (123d / i-123r okanye i-123r / i-123r) inxulumene ne "r / i-123r "idityaniswa ne-trances kwi-Proulsion (123R) , ukunyusa intsebenziswano (umzobo. Ingcaciso kukuba le meko intshabalalo yokubeka intsalela ye-hydrophobic kwindawo ye-hydropholic inokukhupha impembelelo yokuzinzisa i-D123 yentsalela yokuthintela intsebenziswano. I-D171 ye-ciona gutis ci-h1 I-Gating ye-HV1 izitishi ziyaziwa ukuba zenzeke kwiinguqu ezininzi19,20,29,39,44,401.Qiu et Alporication, i-voltage et alforner eshiya i-witnel iqhubekile kodwa ivaliwe , kulandele inguqu eyahlukileyo ebangela iiprotens ekuvuseleni zombini iindlela ze-sundege. I-PRTURTURCTURCY SINGINGY ingqinelana nobukho bamanqaku e-ELTrostatic phakathi koluvo olufakelweyo lwe-D173 ngokusebenzisana kwethu kwi-D123 isebenzisana ngokudibeneyo kunye nesalals ye-Affaphuclic Couplation phakathi kweendawo zokubopha i-GBTA. I-fujiwara et al25 icekise ukuba i-Dimer ye-District ye-Cytoplasmic ye-Cytoplasmis ye-Cytoplasmi ye-S4 ye-S4 ye-S4 .A Imodeli yale ncwadana i-Intertersit isekwe kwi-Cysteine ​​Cross Yonke i-VSD kunye nohlalutyo olusebenzayo lwengingqi edibanisa i-S4 SHDEAR kunye ne-CCD.in ukungabikho kwe-promerane PH ye-Gedmentrane PH GOORMANE, kunye nokuCingiswa kwe-HV1 kufuna ukuba kuvalwe indawo apho itshaneli ivaliwe. Ke ngoko, i-Stal-S4 yoNxibelelwano ye-S4-S4 ibonisa ubungqina bomhlaba we-S4-tures. Amazwe avaliweyo, umbono ohambelana nokufumana i-Mony et al. I-S1 ihamba i-39 ngexesha le-git. Apha, sibonisa ukuba, kwindawo evulekileyo, i-Overcellifor SEPRING ye-S1 HITEXOLY isondele kakhulu ekusombululeni phakathi kwe-s4-s4 ye-S4-S4, ii-S1 ze-S1 zikude kakhulu Ngaphandle komnye nomnye ukunxibelelana ngqo.Ise-20 ° Ukujikeleza kwe-20 ° Ukujikeleza kwe-vsd ye-vsd kwi-Axis kwindiza ye-S4, kudityaniswe nobume be-S1-S1 ngokuguqula Ngeziphumo zethu (umzobo. Nangona i-enzyme ye-enzyme icingelwa ukuba isebenze njengomlinganiswa, isakhiwo sekristale ye-vmd yayo ifakwe kwindawo ye-Dimer. ye-HV1 (FIG. kwaye imibala yendawo iphakamisa ukuba i-VSDS yezi proteni inotyekelo lwangaphakathi ukwenza isiphelo se-S1 sihambahamba. Indima ebalulekileyo ye-HV1 kwi-sperm yeselfowuni yenza ukuba le tekshi ijolise ekuchumeni kolawulo lobudoda.Ukusebenza kwe-HV1 kwi-HV1 ye-HV1 ifunyenwe idityaniswa kunye Ukusinda kwizigulana ngebele le-12 okanye umhlaza we-13 okanye kucingelwa ukuba unegalelo kwi-AgesPhesProes ye-HV1 inokusetyenziswa njenge-Hianidive Agements. Vula i-HV1 i-HV1 i-HV1 i-HV1 i-HV1, ekhokelela ekunyukeni ngokubondayo, inokukhokelela kuphuhliso lwamachiza anobuchule obujolise kujoliso lwe-HV1. I-STE-ehlomezwe i-sugeragessis ye-HV1 ye-HV1 isebenzisa ubugcisa be-PCRCR.in i-HV1NCCIVIV SALTIONAL, i-DV1 ukuya kwi-HV1 ithathelwe indawo yindawo ye-1-11-73 ye-dimer ye-HV1 ye-HV1, I-C-theminus ye-subnit enye inxulumene ne-N-theminus ye-SATGSGSGSGSGSGSGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGSGSGGSGGSGGSGGSGSGGS.I-ENGLMS ye-Enzyment equkethe i-Enzys kunye ne-Enzymes ye-Nhe1 I-T7 MMESSSM UMMACECCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCRCECRCRCRCRCRCRCRCRCRCECCRCY STATE Ukusuka kwinaliti ye-ecocyte.Fung inaliti ye-RNA, iiseli zagcinwa kwi-ND96 i-PMM Naacl, i-2 mm KCL, i-10 mm ye-MM POGCL, i-5 mm ye-ML. . I-2-Gunidino-Benzimifino [1], 2-Guinidino-Banidiazole [2], (i-1], (5-i-5-I-4-I-1-1,3 -thiazol-2-YL I-5-i-carxylates [9] kunye (2-Guinidino-4-Giazol-5] yayivela kwi-Sigma-Aldrich - 7] yayivela kwi-MP Biomedicals.1- [4 - (4-Chlorophenyl) -1,3-thiazol-2-Yl Rianidine [11] kunye ne-1- (3)-(3,4-YL] ukusuka kwi-Matrix yesayensi.Izona ziqokelelo eziphezulu zezona zinto zibalulekileyo kuchithwayo. ubuncinci i-99% icocekile. Ukunqunyanyiswa kwe-2-Amino-4- (i-trifluoromethyl) ye-benzenethiol hydrochloride (1.02 g, 4.5 ML ye-ACDROHNAAMARICE Acid (2.5 NG, 4.5 Mmol), kunye nomxube oqingqiweyo i-curlued kunye ne-stirrous ishukumisa iiyure ezi-4.Imixube yokuphendula yaphathwa gadalala kwaye ifakwe ngokungathamsiyo i-Spoticide ye-10n Homes. I-65 ° C) kwiiyure ezininzi, emva koko iphinde yenzeke ukusuka kwi-Ethyyl Acetate / iPetroleum i-Ether ukunika isiseko esimhlophe (500 mg, 48%); I-MP 221-222 ° C (Ukukhanya 225-226 ° C); I-1h NMR (500 MHZ, i-DMSO-D6): , 1 h = 8.1 hz) .13C NMR (200 MHZ, J = 3.5 HIT), 121.7, 124.6 (Q, J = = 272 hz), 126.1 (Q, J = 272 hz), 13.4 hz), i-138.4. : 261.0419. I-Naphtho [1,2-D] GINAZOL-2-AMG (300 MG, i-MMOL), idityaniswe njengoko ngaphambili ichazwe kwi-oyile ye-oyile kwi-tube yovavanyo oluncinci kwaye I-1.0 ml trig.to i-computer eshushu yongezwa i-hydrochloric acid kwaye umxube ugcinwe kwi-oyile yokuhlambela kangangemizuzu emi-2 ubudala, ngexesha lokuphalazwa kwamanzi. yaphuka ibe ngamaqhezu amancinci kwaye ihlanjwe ngamanzi ukuze inikezele ngokuqinileyo kwe-amorphous etyheli. (38 mg, 10%) MP 246-20) MP 246-250 c; I-1h NMR (500 MHZ, DMO-D6, D2O): δ [i-PPM] = 8.2 H = 8.3 H = H . MHZ, DMSO-D6): δ = 119.9, 122.7, 12, 13, 13, 13, 13, 12.4.7, 128s (M +) +: 24699 , ifunyenwe: 243.0704. I-proton curmares ilinganiswe kwi-osches yangaphakathi nangaphandle ye-Oocytes ebonisa eyahlukileyo esebenzisa i-AXPKATATATATATATATATATATATATE ilawulwa yi-Axlamp ye-Axlamp ye-Axlamp ye-ATLAMETATATATATATATATATATATATATE i-Axlamp10 yeSoftware. ) I-Ethanesfoani e-Ethanesfonis (i-mes), i-30 mm Tetrathylemmoemmon (i-Tea) i-mesylate, 5 mm ye-chloride, i-5 mm ye-geracetic acid (engu-In 'ehlengahlengiswayo I-6.0 ngeti i-tea hydroxide. ) Imvelaphi8.1 (ImvelaphiLAB). Izisombululo eziqukethe ugxininiso olwahlukileyo lwe-HV1 inhibitor kwaye kwezinye iimeko i-10 mm me yaziswa kwindawo yokuhlambela kwi-vanold valt yenkqubo ye-vc-6 ye-PCLAMP.), Eyayidluliselwe kwisoftware yePCLAM ( I-Transictor Logic) Imiqondiso.Izilingo zokuvavanywa kwe-profusion zenziwa kusetyenziswa ipensile ye-temputer ye-tube. +120 mv Depolaries Exprelimes yenziwa njengoko bekuchaziwe ngaphambili18,20.imigaqo ye-MV emva kweMiporsulies kwii-Vills ezivela kwi-NJE +120 mv ngaphandle kwe-pulse ephambili isetyenziselwe ukulungisa ukubola okwangoku kwe-18 .Igrafu ye-GV ihambelana ne-Boltzmann Equation: I-KD (i-KD) yokudityaniswa kweziteshi kunye ne-inhibitority (itheyibhile ye-1) igqityiwe ngokuxhomekeka kwe-inbest Ndikunye ne-gesi equation: Apho [ndi] ukugxininiswa kothintelo lwe-I kwaye h yinduli i-coonderand.com