Mukammallik klasteri Frankfurt makromolekulyar komplekslari - Cluster of Excellence Frankfurt Macromolecular Complexes

The Frankfurtning mukammallik klasteri "Makromolekulyar komplekslar" (CEF) tomonidan 2006 yilda tashkil etilgan Gyote universiteti Frankfurt bilan birga Maks Plank nomidagi biofizika instituti va Maks Plank nomidagi miya tadqiqotlari instituti kontekstida Germaniya universitetlarining mukammallik tashabbusi. Tomonidan moliyalashtirish Deutsche Forschungsgemeinschaft (DFG) 2019 yil oktyabr oyida tugaydi. CEF bo'yicha uzoq yillik hamkorlik tadqiqotlari natijasida o'sdi membrana oqsillari va RNK molekulalar va boshqa olimlarni Frankfurt / Mainga jalb qilish orqali ushbu sohalarda tadqiqot ishlarini kuchaytirdi. CEF 45 ga yaqin tadqiqot guruhlarini birlashtirdi, ularning aksariyati Riedberg kampusida joylashgan Frankfurt / Main. CEF tashkil etdi Buchmann Molekulyar Hayot Fanlar Instituti (BMLS).

Maqsadlar

CEF olimlari yirik makromolekulyar komplekslarning, xususan membrana oqsillari va ularning birikmalarining, tarkibiga kiradigan komplekslarning tuzilishi va funktsiyasini o'rganishga kirishdilar. signal uzatish va sifat nazorati va RNK-oqsil komplekslari.

Tadqiqot

CEFda makromolekulyar komplekslarning muhim tuzilmalari aniqlandi. Muhim membrana komplekslariga misol qilib atomlarning tuzilishi kiradi murakkab I va ATP sintezi ning mitoxondriyal nafas olish zanjiri va antigenni qayta ishlash bilan bog'liq bo'lgan transportyor (TAP). Bo'yicha tadqiqotlar RNK tuzilishi va funktsiyasi haroratni sezishning tartibga solish tamoyillarini aniqlashga olib keldi riboswitches, ning tuzilish-funktsiya munosabatlari RNK polimeraza I funktsiyalari mikroRNKlar va mexanizmlari rRNK davomida pishib etish va quyi oqim jarayonlari ribosoma biogenezi va qayta ishlash. Masalan, CEF olimlari retseptorlarini aniqladilar hamma joyda zanjirlar proteazom, chiziqli ubikuitin zanjirlarining rolini ochib berdi va tartibga soluvchi makromolekulalarni tavsifladi mitofagiya, ksenofagiya va ER-fagiya. Ular rolini ajratib ko'rsatdilar sumoylyatsiya yilda ribosoma sifat nazorati va genetik sifat nazorati jarayonini xarakterladi oositlar. Ushbu uchta tadqiqot sohasidagi sa'y-harakatlar makromolekulyar komplekslarni loyihalash yoki qayta dasturlash yondashuvlari va allaqachon kuchli tajribani kengaytirish uchun ishlab chiqilgan yangi usullar bilan birga olib borildi. CEF olimlari tamoyillarini o'rnatdilar va rivojlantirdilar optogenetika shuningdek, yorug'likni tartibga solish uchun biokimyoviy usullar. Shuningdek, ular makromolekulalarni strukturaviy va funktsional tavsiflash uchun biofizik usullarni ishlab chiqdilar. Masalan, hujayra ichidagi dasturlar uchun ishlab chiqilgan nurli o'zgaruvchan molekulalar va RNK katlamasini o'rganish uchun vaqtni aniqlash usullari. Yorug'lik varag'i lyuminestsentsiya mikroskopi rivojlanish va LILBIDni kuzatish uchun mass-spektrometriya membrana komplekslarini tahlil qilish uchun yaxshilandi. PELDOR-EPR hujayra ichidagi o'lchovlarni amalga oshirishga imkon beradigan rezolyutsiya asosida ishlab chiqilgan bo'lib, Klaster bir qator dasturlar, shuningdek, seminarlar, xalqaro konferentsiyalar va ma'ruzalar seriyalari orqali ilmiy almashinuvni rivojlantirdi. Optogenetika va yorug'lik varaqlari lyuminestsent mikroskopi " Yil "fanlarning barcha sohalarida va muhandislik sohasida ilmiy-tadqiqot jurnalida chop etilgan Tabiat usullari navbati bilan 2010 va 2014 yillarda.[1][2]

CEFning beshta tadqiqot yo'nalishi quyidagilarni o'z ichiga olgan: (A) Membranadagi komplekslarning tuzilishi, mexanizmlari va dinamikasi, (B) Sifatni nazorat qilish va signalizatsiya qilishda makromolekulyar komplekslarning tarkibi va dinamikasi, (C) Ribonuklein kislota-oqsil-komplekslarning dinamikasi, ( D) makromolekulyar komplekslarni loyihalash va (E) makromolekulyar komplekslarni o'rganish usullari.

CEF tadqiqot yo'nalishi A - membranadagi komplekslarning tuzilishi, mexanizmlari va dinamikasi

Biologik membranalar Hayotiy jarayonlarda juda muhim rol o'ynaydi, chunki hujayraning yashashi, o'sishi va javob berishi uchun zarur bo'lgan barcha narsalar u orqali o'tishi yoki harakat qilishi kerak. uyali nafas olish va fotosintez membranalarda sodir bo'ladi, har qanday sezgir stimul va miyada axborotni qayta ishlash ular vositachiligida bo'ladi. Ushbu turli xil harakatlar majmuasi juda ko'p sonli turli xil tomonidan amalga oshiriladi membrana oqsillari. Hujayra membranasining gavjum sharoitida ko'pgina membrana oqsillari turli xil vazifalarini bajarish uchun murakkab dinamik yig'ilishlarga birlashadi. Shu sababli va ular membrananing lipidli ikki qatlamiga singib ketganligi sababli, aksariyat membrana oqsillarini o'rganish qiyin va ularning funktsiyalari ko'pincha hal qilib bo'lmaydigan bo'lgan. CEF olimlari ushbu muammolarning bir qismini engib o'tish uchun yangi ishlarni amalga oshirdilar va bir qator muhim yirik majmualarning tuzilishi, mexanizmlari va tartibga solinishiga katta hissa qo'shdilar, shu jumladan. nafas olish kompleksi I,[3][4]aylanadigan ATPazalar,[5][6][7][8] superkompleks I1III2IV1[9],[10] sitokrom cbb3 oksidaza,[11]sitoxrom bd oksidaz,[12] sulfid: xinon oksidoreduktaza,[13]qo'ziqorinli TOM yadro kompleksi,[14]bakteriyalarning ikki teshikli K + qabul qilish tizimi KtrAB,[15]Na + ga bog'liq bo'lmagan karnitin / butirobetain antiporter CaiT,[16] betain / Na + muxlislari BetP,[17]ko'p dori effekti tashuvchisi AcrB[18][19]va shaperone va tahrirlash TAPBPR – MHC I kompleksi[20]va inson MHC-I peptidlarni yuklash kompleksi.[21] TAPda antigenik peptidni tanib olish DNP tomonidan takomillashtirilgan qattiq holatdagi NMR spektroskopiyasi bilan hal qilindi.[22] Dipolyar EPR spektroskopiyasi yordamida inson antigen tashuvchisi ortolog TmrABdagi konformatsion birikma va trans-inhibisyon hal qilindi.[23]Membrana oqsillarini 3D tuzilishini aniqlashdagi yutuqlar Rentgenologik kristallografiya va kriyo elektron mikroskopi magnit-rezonans usullari bilan chuqur mexanistik tadqiqotlar uchun talab va imkoniyatlarni yaratdi. Membranadagi oqsillarga xos bo'lgan muammolar tufayli, taraqqiyot metodlarni ishlab chiqishda birinchi o'rinda turadigan texnika mavjudligiga bog'liq. Ayniqsa, qattiq holatli (MAS) NMR membranani oqsillarini to'g'ridan-to'g'ri ikki qatlamli muhitda tekshirish orqali "statik" tuzilmalar va biokimyoviy ma'lumotlar orasidagi farqni ko'paytirishga imkon beradi. Bunday tajribalar qiyin va yutuqlarga faqat sezgirlikni kuchaytirish uchun dinamik yadro polarizatsiyasi va spektral o'lchamlari uchun juda yuqori magnit maydonlar tufayli erishish mumkin edi. CEF olimlari ABC transportyorlarining katalitik mexanizmi to'g'risida yangi tushunchalar berishga muvaffaq bo'lishdi. Haqiqiy vaqtda 31P-MAS-NMR asosida ular homodimerik lipid A ekanligini aniqladilar flippaza MsbA ATP gidrolizidan tashqari teskari adenilat kinaza o'xshash reaktsiyani katalizatsiyalashga qodir.[24]Bundan tashqari, ATP gidroliz tsikli ABC tashuvchisi LmrA uchastkaga yo'naltirilgan spin yorlig'i va impulsli elektron-elektron juft rezonans (PELDOR / DEER) spektroskopiyasi bilan tekshirildi.[25]Ikkilamchi ko'p dori effekti nasosi EmrE dan E. coli 31P- va DNP kuchaytirilgan qattiq holatdagi NMR bilan keng o'rganilgan.[26]Shuningdek, bir qator fotoreseptorlar kabi mikrobial rodopsinlar trans-membranali transport jarayonlarida ishtirok etadi. Masalan, CEF tarkibidagi guruhlar bo'yicha pentamerik nurli proton pompasi bo'lgan proteorhodopsinning strukturaviy va funktsional tavsifiga katta hissa qo'shildi.[27][28].[29]CEF tadqiqotchilari ishlab chiqdilar mass-spektrometriya katta membranali oqsil komplekslari uchun mos keladigan yondashuvlar. Lazer ta'sirida suyuqlik nurlari / boncuklar ionlarining desorbsion mass-spektrometriyasi (LILBID) 1 MDa va undan ortiq butun membrana oqsil komplekslarini ommaviy tahlil qilishga imkon beradi.[30] CEF olimlari guruhi insonning subtipi selektivligi mexanizmini hal qildi bradikinin retseptorlari ularning peptid agonistlari uchun DNP-yaxshilangan integratsiya qilish orqali qattiq holatdagi yadro magnit-rezonansi rivojlangan molekulyar modellashtirish va joylashtirish bilan[31]

CEF tadqiqot yo'nalishi B - Sifatni nazorat qilish va signalizatsiya qilishda makromolekulyar komplekslarning tarkibi va dinamikasi

Uyali sifat nazorati dasturlarini boshqaruvchi signalizatsiya komplekslarining funktsiyalari va tarkibiy tarkibini tavsiflash CEF tadqiqotlarining asosiy mavzularidan biri edi. Oqsillar yakka vujudga aylanadi degan fikr, signalosomalar sifatida izohlangan multimerik eruvchan komplekslarning dinamik qayta tashkil etilishi hujayradagi signallarning tarqalishi uchun juda muhimdir degan tushunchaga almashtirildi. Ushbu komplekslarning faoliyatini tartibga solish ularning dinamik tarkibi bilan ham erishiladi tarjimadan keyingi modifikatsiyalar (PTM) oqsillar. Ushbu modifikatsiyani tan oladigan domenlar hujayraning o'zlarining mikro muhitidagi o'zgarishlarga javob berishida hal qiluvchi rol o'ynaydi. CEF tomonidan bir nechta signalizatsiya yo'llarini tavsiflash va ularni PTMlar tomonidan tartibga solish bo'yicha muhim yutuqlarga erishildi, shu jumladan. hamma joyda o'xshashlik, fosforillanish va atsetilatsiya. CEF-dagi tadqiqotlarning asosiy yo'nalishi avtofagik va ubikuitin / proteazomal yo'llar uchun asos bo'lgan oqsil sifatini boshqarish mexanizmlari, nuqsonli yoki ortiqcha oqsillarni, komplekslarni va organoidlarni parchalash uchun ishlatiladigan ikkita uyali tizimga qaratilgan. CEF tadqiqotlarining qo'shimcha markazlari genetik sifat nazorati edi oositlar va epiteliy ildiz hujayralari tomonidan p53 oqsil va regulyatsiyasi va tomonidan kinazlar.

Avtofagiya bo'yicha tadqiqotlar

Selektiv paytida avtofagiya, yuk degradatsiyasi uchun maxsus mo'ljallangan va selektivlikni tartibga soluvchi alohida yuk retseptorlari tavsiflangan. Ushbu jarayonni avtofagiya retseptorlari o'z yuklarini maxsus tanib olishlari va bog'lashlari va fagoforga etkazishlari bilan osonlashtiradi. Odamlarda olti xil LC3 / mavjudGABARAP tug'ilishni bog'lash orqali markaziy rol o'ynaydigan oqsillar avtofagosoma yutish jarayonini osonlashtiradigan membranalar va yuk bilan to'ldirilgan autofagiya retseptorlari, ba'zida vositachilik qiladi yoki qo'shimcha adapter oqsillari tomonidan qo'llab-quvvatlanadi.[32]CEF olimlari GABARAP oqsillari nafaqat autofagiyada, balki uvikitinga bog'liq degradatsiyaga ham aloqadorligini ko'rsatdilar. TIAM1.[33]Hujayralar hujayra ichidagi patogenlar bilan qanday kurashish va hujayra ichidagi bakteriyalar bu qarshi choralardan qanday qochishga harakat qilishlari bo'yicha yutuqlarga erishildi. Kinaz Tbk1 optineuringa asoslangan vositachilik uchun muhim deb topildi ksenofagiya yuqtirilgan hujayralardan bakteriyalarni olib tashlash.[34]Mass-spektrometriyadan foydalanib, ning hamma joyda mavjud bo'lgan global tahlili Salmonella infektsiyalangan hujayralar amalga oshirildi, bu CEF olimlariga bakteriyalarning aniq maqsadlarini aniqlashga imkon berdi ligazlar hujayra ichiga ajratilgan sitoplazma patogenlar tomonidan.[35]CEF olimlari, shuningdek, fosforibozil bilan bog'langan yangi turdagi molekulyar mexanizmni ochib berishdi serin qo'zg'atuvchining SdeA effektori bilan hamma joyda tarqalishi Legionella, bu kanonikdan juda farq qiladi lizin -bubikitatsiya mexanizmi.[36][37]Ular bundan tashqari yana bir effektorini ko'rsatdi Legionella bakteriyalar, SidJ, xamirturush va sutemizuvchilar hujayralarida SidE ning toksikligiga qarshi.[38]Ommaviy spektrometriya tahlili shuni ko'rsatdiki, SidJ SdeA ning mono-ADP ribosil transferaza domenidagi katalitik glutamatni o'zgartiradigan glutamilaza bo'lib, shu bilan SdeA ning ubikuitin ligaz faolligini bloklaydi. Bundan tashqari, ular retikulontip oqsillari ERga xos autofagiya retseptorlari vazifasini bajarishini aniqladilar va ularning membrana egriligiga ta'sirini simulyatsiya qildilar.[39][40]

Joylashuv

Avtofagiya yo'li orqali yoki degradatsiyaga uchragan oqsillarni markirovka qilishda uvikitinatsiya markaziy rol o'ynaydi. proteazom. CEFning bir nechta guruhlari ubikuitin signalizatsiyasi nafaqat degradatsiya signali sifatida ishlatilishini, balki boshqa bir qator uyali jarayonlarda qanday ishtirok etishini tushunishda yutuqlarga o'z hissalarini qo'shdilar.[41][42] [43][44][45][46]

p63

Bo'yicha tadqiqotlar TP63, shuningdek, p63 nomi bilan ham tanilgan bu oqsil qatlamli epiteliya to'qimalarining ko'payishi va differentsiatsiyasi uchun hamda ayol jinsiy hujayralarida genetik sifatni kuzatish uchun muhim rol o'ynaydi. CEF olimlari tomonidan o'tkazilgan tadqiqotlar shuni ko'rsatdiki, p63 ning o'ziga xos izoformasi mayoz I profazasida hibsga olingan ibtidoiy oositlarda yuqori darajada namoyon bo'ladi. Ushbu izoform yopiq, harakatsiz va faqat dimerik konformatsiyani qabul qiladi, unda ikkalasi ham DNK bilan o'zaro ta'sirida va transkripsiya apparati sezilarli darajada kamayadi[47]Tormozlanish olligomerizatsiya domenining tetramerizatsiya interfeysini olti qatorli anti-parallel beta-varaq bilan blokirovkalash orqali amalga oshiriladi.[48] Aktivizatsiya fosforillanishni talab qiladi va bahorda qaytarib bo'lmaydigan faollashtirish mexanizmiga amal qiladi.[49] Ushbu kashfiyotlar davomida oositlarni saqlab qolish uchun terapiyani ishlab chiqish imkoniyatini ochadi kimyoviy terapiya bu ayol saraton kasalligida odatda bepushtlik va erta boshlanishiga olib keladi menopauza. CEF olimlari shuningdek anamellofaron-ektodermal displazi-yoriq lab / tanglay sindromi, SAM domenidagi yoki C-terminalidagi mutatsiyalarga asoslangan teri eroziyalari, og'zaki yoriqlar anormalliklari va eritilgan ko'z qovoqlari kasalligi sabab bo'lgan molekulyar mexanizmni aniqlashda yordam berishdi. p63 ning.[50]Shish paydo bo'lishida ishtirok etgan komplekslar bir qancha CEF guruhlari, shu jumladan leykemogen AF4-MLL termoyadroviy oqsillari tomonidan o'rganilgan.[51]va turli xil shakllarni boshlash va nozik sozlash uchun muhim bo'lgan RIP1 o'z ichiga olgan sitosolik komplekslar hujayralar o'limi, ya'ni apoptoz va nekroptoz[52][53]

SGC Frankfurt

Gyote universiteti a'zosi bo'ldi Strukturaviy Genomika konsortsiumi (SGC) 2017 yilda muhim oqsillarning tuzilishini aniqlashga va biologik makromolekulalarning inhibitorlari va zondlarini ishlab chiqishga bag'ishlangan xalqaro konsortsium va davlat-xususiy sherikligi funktsional tekshiruvlarda foydalanishi mumkin. Gyote universiteti, shuningdek, SGC tomonidan sovg'a qilingan problar dasturi uchun uy va ma'lumot markaziga aylandi, bu esa kichik molekulalarni endi sanoat tomonidan ta'qib qilinmaydi, chunki giyohvand moddalari butun dunyo bo'ylab tadqiqotchilarga erkin taqdim etiladi.[54]). CEF olimlari ishlab chiqdilar bromodomain inhibitörleri bu atsetil-lizin modifikatsiyasini bog'laydigan domenlarning funktsiyasini o'rganish uchun ishlatilishi mumkin. Zondlar to'plami o'ziga xos bromodomanlar uchun vositalar sifatida tavsiflangan va tasdiqlangan[55]

Membranadagi eruvchan domenlar bilan o'zaro ta'sir

CEF buni ko'rsatdi qon tomir endotelial o'sish omili retseptorlari -2 ichki holatga keltirilishi kerak va uning assotsiatsiyasi tomonidan tartibga solinadi efrin Endotelial hujayralardagi Bs.[56] EphrinBs darajasi nazorat qilish uchun ham muhim ekanligi aniqlandi AMPA retseptorlari sinaptik membranada.[57]Quyruq bilan biriktirilgan oqsillarni membranaga kiritish mexanizmi, eruvchan Get3 oqsilining membrana bilan bog'langan Get1 va Get2 retseptorlari sitoplazmatik domenlari bilan o'zaro ta'sirini tizimli va biokimyoviy tavsiflash orqali o'rganildi.[58]

CEF tadqiqot yo'nalishi C - ribonuklein kislota-oqsil komplekslarining dinamikasi

Ko'pgina kashfiyotlar, shu jumladan kodlashning bir nechta sinflarini aniqlash RNKlar va tartibga soluvchi RNK elementlari RNK funktsiyasi nuqtai nazarini passiv ma'lumot tashuvchisidan faol uyali komponentga kengaytirdi. Uning strukturaviy va funktsional tavsifi molekulyar o'zaro ta'sirni va unga bog'liq bo'lgan dinamikani tushunish uchun talab qilinadi.

RNK elementlarining tarkibiy tavsifi va ularning dinamikasi

RNK tuzilmalarini yuqori aniqlikdagi NMR asosidagi tahlilining kombinatsiyasi[59][60]va aniq konformatsiyalarni qafas qilish yo'li bilan RNKlarning ligand ta'sirida qayta katlamasi [61]impuls bilan birga elektron paramagnitik rezonans spin-markirovkadan so'ng bazaga xos usullar (PELDOR)[62] [63][64]va ultrafast lazer spektroskopiyasi RNK dinamikasining [65][66]bir nechta RNKlarning strukturaviy dinamikasini tavsiflashga olib keldi.CEF olimlari adenin sezgirligini boshqarish mexanizmi riboswitch odamning patogen bakteriyasi Vibrio vulnificus uch holatli barqaror konformatsiyani o'z ichiga olganligi bilan ikki holatli kalit mexanizmidan farq qiladi. Ushbu tarjima adeninni sezuvchi riboswitch haroratni qoplaydigan regulyatorli RNK elementining birinchi namunasini namoyish etdi.[67]Ning tarkibi va tuzilishi OIV TAR RNK-Ligand kompleksi LILBID va NMR [68] ,[69]RNKdagi peptidlarni bog'lash joylarining murakkabligini tavsiflashga olib keladi, shuningdek, guaninni sezuvchi riboswitch-aptamer domeni Bacillus subtilis xpt-pbuX operon, Diesel-Alderaza ribozimlar[70]RNK asosidagi termometr,[71]va N1–ribostamitsin murakkab[72]ECF olimlari shuningdek, guaninni sezuvchi xpt-pbuX riboswitch uchun B. subtilis, to'liq metrajli transkriptlarning konformatsiyasi statik: u faqat funktsional holatni to'ldiradi, lekin ligandning mavjudligidan yoki yo'qligidan qat'i nazar, holatga o'tolmaydi. Faqat transkripsiya stavkalari va ligandni bog'lashning bir-biriga mos kelishi transkripsiyaning qidiruv vositalariga ligandga bog'liq bo'lgan konformatsion qayta to'ldirishga imkon beradi[73](Steinert va boshq., 2017).

Eukaryotlarda ribosoma biogenezida ishtirok etadigan komponentlar

Bilan hamkorlikda CEF olimlari Maks Plank nomidagi biofizik kimyo instituti ingl RNK polimeraza Men (Pol I) faol transkriptsiya jarayonida ribosoma genlar hujayra muhitida va uning tuzilishini nuklein kislotalar bilan va ularsiz 3,8 Å piksellar bilan hal qildi krio-EM.[74]Ularning tuzilmalari tartibga solishni tushuntirdi transkripsiya qisqargan va kengaytirilgan polimeraza konformatsiyalari mos ravishda faol va harakatsiz holatlar bilan bog'liq bo'lgan cho'zish.

Bir nechta CEF guruhlari o'rtasidagi hamkorlik natijasida molekulyar xususiyat aniqlandi Bowen-Konradi sindromi ribosoma biogenez omilining kasallik keltirib chiqaruvchi nuqta mutatsiyasini namoyish etish orqali Nep1 uning nukleolyar lokalizatsiyasi va RNK bilan bog'lanishini susaytiradi.[75] [76]Boshqa bir ishda Edinburg universiteti bilan hamkorlikda RNKning o'zaro bog'lanishi va cDNA (CRAC) tahlili orqali RNK helikaza Prp43 tahlil qilindi va ribozom biogenezidagi ushbu fermentning funktsional rollari to'g'risida dastlabki tushunchalar berildi.[77]CEF olimlari o'simlikdagi o'ziga xos ribosoma biogenez omillarini ham aniqladilar A. taliana muhim funktsiyasi bilan rRNK qayta ishlash[78]va ekanligini ko'rsatdi 60S -LSG1-2 uchun assotsiatsiyalangan ribosoma biogenez faktori zarur 40S pishib etish A. taliana.[79]

RNK-modifikatsiyalovchi fermentlar va RNK molekulalarining tarqalishi

Eukaryotik hujayralardagi tabiiy muhitdagi RNPlarning dinamikasi yuqori aniqlikdagi mikroskop yordamida ingl.[80]Adenozin-inozin (A-I) RNK tahriri, bu katalizlanadi adenozin deaminaz RNK (ADAR) fermentlariga ta'sir qilish, RNK metabolizmini epitranskriptomik boshqarishda muhim ahamiyatga ega. Katepsin S (CTSS) mRNK, bu bilan bog'liq bo'lgan sistein proteazini kodlaydi angiogenez va ateroskleroz, inson tomonidan yuqori darajada tahrirlanganligi ko'rsatildi endotelial hujayralar .[81]A-I RNK tahriri, aterosklerozda katepsin S ekspressionini boshqaradi, bu esa transkripsiyadan keyingi HuR vositachiligiga bog'liq.

dan mRNA eksporti yadro sitoplazmasiga juda tartibga solingan qadam gen ekspressioni. CEF olimlari a'zolarni baholadilar SR oqsili moslashtiruvchi sifatida harakat qilish potentsiali uchun oila (SRSF1-7) yadroviy eksport faktor 1 (NXF1) va shu bilan er-xotin mRNKgacha qayta ishlash mRNA eksportiga.[82]Ular> 1000 ta endogen mRNK yadro eksporti uchun alohida SR oqsillarini talab qilishini aniqladilar jonli ravishda. Mexanizmga murojaat qilish uchun, transkriptom NXF1 va SRSF1-7 ning keng tarqalgan RNK-biriktiruvchi profillari parallel ravishda individual nukleotid rezolyutsiyasi bilan o'zaro bog'liqlik va immunoprecipitatsiya bilan aniqlandi (iCLIP ). SRSF3 So'nggi ekzonslarda NXF1 tomonidan RNK bilan bog'lanishning ketma-ketligi o'ziga xosligini keltirib chiqaradigan eng kuchli NXF1 adapteri sifatida paydo bo'ldi. Ko'p sonli inson kasalliklari keng tarqalgan regulyatsiya bilan tavsiflanadi RNK bilan bog'langan oqsillar (RBP) va katta darajada o'zgartirilgan transkriptom naqshlar. CEF olimlari tadqiqotchilar bilan hamkorlikda transkriptomiya miqyosida posttranskripsiyani tartibga solish mexanizmlarini o'rganish uchun hisoblash usullaridan foydalanganlar. Mayns IMB.[83]

Kodlamaydigan RNKlar

CEF olimlari, shuningdek, yangi | kodlashsiz RNKlar]] ta'sirini tekshirdilar uzun bo'lmagan kodlash RNKlari (lncRNAs) va mikroRNKlar (miRNA), hujayra funktsiyasi to'g'risida. miRNAlar mRNK bilan bog'lanish va ularning tarjimasini oldini olish orqali gen ekspressionini tartibga soladi. CEF Fokus loyihalaridan biri neyronlarda faollikka bog'liq fazoviy-lokalizatsiya qilingan miRNK kamolotini kuzatishda muvaffaqiyatga erishdi. dendritlar.[84]MiRNA ning mahalliy pishishi oqsil sintezining mahalliy qisqarishi bilan bog'liqligi aniqlandi, natijada mahalliy miRNK kamolotining maqsadli gen ekspressionini mahalliy va vaqtinchalik aniqlik bilan modulyatsiya qilishi mumkin. LncRNA Meg3 endotelial hujayraning qarishini boshqarishi aniqlandi va uning inhibatsiyasi endotelial hujayra funktsiyasining qarish vositachiligida buzilishini qutqarish uchun potentsial terapevtik strategiya bo'lib xizmat qilishi mumkin.[85] LncRNA MALAT1 endotelial hujayra faoliyatini va tomirlar o'sishini tartibga solishi aniqlandi.[86]va tartibga solish orqali aterosklerozdan himoya qiladi yallig'lanish.[87]

CEF tadqiqot yo'nalishi D - makromolekulyar komplekslarni loyihalash

CEF-dagi ishlarning asosiy yo'nalishi metodlarni ishlab chiqish va ulardan foydalanish va o'rganishdir oqsillar hujayra va molekulyar funktsiyalarni yorug'lik bilan modulyatsiya qilishga imkon beradigan. Sohasida optogenetika, nazorat qilish membrana potentsiali va hujayra ichidagi signalizatsiya yilda neyronlar va boshqa hujayralarga fotosensor oqsillarini ekspressioni orqali erishiladi, aksariyat hollarda mikrob kelib chiqishi, masalan. ion kanallari yoki nasoslar, shuningdek nur bilan faollashtirilgan fermentlar. Optokimyoviy yondashuvlar, aksincha, biologik to'qimalarda yorug'lik ta'siriga erishish uchun kimyoviy ishlab chiqarilgan molekulalardan foydalanadi.

Optogenetika

Optogenetikaning kelib chiqishi Bamberg guruhining ishida Biofizika MPI buni kim ko'rsatdi Frankfurtda kanalrhodopsin-2 (ChR2) - bu ifoda etilgan hujayralarni depolyarizatsiyalashga qodir bo'lgan nurli kation kanali.[88][89]CEF davrida Bamberg laboratoriyasi ushbu sohada ishlashni davom ettirdi va bir nechta seminal maqolalarga hissa qo'shdi, masalan. xarakteristikasi bo'yicha[90] [91][92] shuningdek, ChR2 ni turli xil xususiyatlarga ega optogenetik vositalarga muhandislik qilish bo'yicha.[93]Depolarizatsiya uchun ChR2 ning birinchi ishlatilishi sutemizuvchi Frankfurtda birinchi ChR2-transgen hayvonning hujayralari va avlodlari sodir bo'lgan. Gottschalk laboratoriyasida engil dvigatelli Cl-nasosi bo'lgan ChR2 ishlab chiqarildi halorhodopsin va boshqalar rodopsinlar nematodaning asab tizimiga C. elegans, bitta neyronlarni rag'batlantirish va ularning funktsiyalarini xulq-atvor natijasi bilan bog'lash[94][95].[96]Bundan tashqari, ular keyin sinaptik uzatishni o'rganishdi fotostimulyatsiya, ChR2 va fotoaktiv yordamida adenil siklaza (PAC), bilan birgalikda elektrofiziologiya va elektron mikroskopi[97],[98]blokirovka qilish uchun o'zgartirilgan yoki yangi xususiyatlarga ega optogenetik vositalarni taqdim etdi sinaptik uzatish yoki manipulyatsiyasi uchun tsiklik GMP[99][100].[101]Bir nechta CEF guruhlari nafaqat turli xil vaqt o'lchovlarida ChR2 fotosiklini ochish uchun kuchlarni birlashtirdilar[102]Juelich tadqiqot markazi bilan hamkorlikda ChR2 tomonidan ion o'tkazuvchanligi bo'yicha tarkibiy tushunchalar taqdim etildi.[103]Shuningdek, ular ion o'tkazuvchanligi o'zgargan bir nechta mutant ChR2 versiyalarini yaratdilar (masalan, Ca ortdi2+- "CatCh" da o'tkazuvchanlik, Ca2+ optogenetik vositalar qutisiga juda foydali qo'shimchalarni ifodalovchi kanalrhodopsin) yoki kinetikani tashish.[104]2015 yilda CEF olimlari birinchisini taqdim etdilar NMR ChR2 retinal kofaktorining tarkibiy detallarini hal qilgan tadqiqot. Ushbu tadqiqot faqat chunki mumkin edi DNP (gibrid usulni bog'lash EPR bilan qattiq holatdagi NMR spektroskopiyasi ) metastabil qidiruv mahsulotlarni aniqlash uchun sezgirlikni 60 baravar oshirdi. Shu tarzda, qorong'i holatdagi eksklyuziv trans-retinal konformatsiya uchun birinchi aniq dalillar keltirildi va yangi foto intermediat aniqlanishi mumkin edi. Tadqiqot shuni ko'rsatdiki, DNP bilan yaxshilangan qattiq holatdagi NMR rentgen asosidagi strukturani tahlil qilish va funktsional tadqiqotlar orasidagi yuqori aniqlikdagi molekulyar rasmga bo'lgan farqni ko'paytirishning asosiy usuli hisoblanadi.[105]

Asta-sekin rhodopsinlarning funktsiyalari va tarqalishining keng spektriga ega ekanligi aniqlandi hayot fitosi. Yangi rodopsinlar bilan ular ettita strukturaviy iskala saqlanib, juda ko'p qirrali oqsillar oilasini namoyish etishlari kuzatildi. transmembranli vertolyotlar retina bilan xromofor konservatsiyaga bog'langan lizin.[106]CEF olimlari mikrobial rodopsinlarning tuzilishi bilan bir qatorda funktsiyalarini o'rganishdi. Ulardan biri proteorhodopsin, sayyoramizdagi eng ko'p uchraydigan retinaga asoslangan fotoreseptor bo'lgan dengiz mikroblarida uchraydi. Proteorhodopsinlarning variantlari yuqori darajadagi atrof-muhitga moslashishni namoyish etadi, chunki ularning ranglari mavjud yorug'likning optimal to'lqin uzunligiga moslangan.[107][108][109][110][111]

CEF olimlari Germaniyaning boshqa universitetlari hamkasblari bilan birgalikda rodopsin optogenetik vositalarining funktsional xususiyatlarini, ya'ni retinal xromofor modifikatsiyasini o'zgartirish uchun yangi yondashuvni ishlab chiqdilar. Sintetik retinal analoglar ChR2 yoki boshqa rodopsin vositalariga kiritildi C. elegans, Drosophila va optogenetik aktuatorlarning yorug'lik sezuvchanligini, foto tsikl kinetikasini va rang spektrini o'zgartirish uchun inson hujayralari.[112]Ular shuningdek, qattiq nur bilan boshqariladigan guanil-siklazani o'rnatdilar opsin Tezlik bilan yorug'lik bilan ishlaydigan cGMP o'sishini ta'minlaydigan CyclOp.[113]CEF olimlari tahlil qilish uchun optogenetik vositalardan ham foydalanishgan asab zanjirlari va ular xatti-harakatlarni qanday boshqarishi.[114][115][116]

Optokimyoviy yondashuvlar

Boshqarmoq oqsillar va nuklein kislotalar engil CEF olimlari tomonidan bir qator ishlab chiqilgan va qo'llanilgan fotosuratlar boshqa, ribonukleozidlar va nuklein kislotalar, RNK aptamerlari va "mayoqlar".[117][118][119][120][121]Shuningdek, ular uchun yondashuvni ishlab chiqdilar kimyoviy-fermentativ sintez biofizik tadqiqotlar uchun maxsus o'zgartirilgan RNK pozitsiyasi, shu jumladan nurni boshqarish.[122] Bundan tashqari, DNK nanoimarkituralarining nur bilan faollashtiriladigan o'zaro ta'siri, nuklein kislotalardagi nurga bog'liq konformatsion o'zgarishlar, nurga bog'liq RNK aralashuvi va nurga bog'liq transkripsiyasi amalga oshirildi.[123][124]Nuklein kislotalar uchun to'lqin uzunligini tanlab nurni qo'zg'atuvchi vosita yaratildi[125] shuningdek, uch o'lchovli boshqarish DNKning gibridizatsiyasi tomonidan ortogonal ikki rangli ikki fotonli kassa.[126] CEF olimlari uch o'lchovli fotoreliz uchun qizil siljigan ikki fotonli qafas guruhini ishlab chiqdilar.[127] Shuningdek, ular molekulalararo va konformatsion jihatdan yaxshi aniqlangan DNK hosil bo'lishiga imkon beruvchi minimal o'zgaruvchan modulni ishlab chiqdilar. G ‐ to'rtburchak fotosurat bilan jihozlangan struktura azobenzol orqa miya tuzilishining bir qismi sifatida qoldiq.[128] Muhimi, shuningdek, an induktorli lyuminestsent prob neyronlarda faollikka bog'liq bo'lgan fazoviy lokalizatsiya qilingan miRNK kamolotini aniqlashga imkon berdi dendritlar.[129]Yorug'lik induktsiyasidan foydalanish antimiRs, CEF olimlari, shuningdek, mahalliy cheklangan maqsadni tekshirdilar miRNA faoliyatida terapevtik foyda bor diabetik jarohatni davolash va yorug'lik terapevtik jihatdan faol antimirlarni mahalliy darajada faollashtirish uchun ishlatilishini aniqladi jonli ravishda.[130]

Uchun yangi qurilish tamoyillari DNK-nanoimarkitekturalari CEF-da tashkil etilgan[131] [132][133]Shuningdek, yangi RNK riboswitches kichik bilan tetiklanadigan dizaynlashtirilgan metabolitlar, ekzogen molekulalar yoki harorat o'zgarishi, shuningdek aptamerlar yoki o'z-o'zini ajratish ribozimlar, bu gen ekspressionini boshqarish uchun ishlatilishi mumkin jonli ravishda.[134]Makromolekulalarni manipulyatsiya qilish uchun nano-miqyosda yanada qulayroq qilish uchun CEF makromolekulyar komplekslarni ikki o'lchovda juda yuqori aniqlikda tashkil qilish uchun qo'llaniladigan usullarni, shuningdek kichik sintetik darvozabonlar va biomolekulyar o'zaro ta'sirlarni boshqarish uchun yangi "yorug'lik kalitlari" va makromolekulyar komplekslarni yig'ishni ishlab chiqdi.[135] [136][137] [138][139] Ikki fotonli faollashuv orqali uch o'lchovli oqsil tarmoqlarini yig'ish usuli ishlab chiqildi.[140]CEF olimlari, shuningdek, optik boshqaruvga erishdilar antigen sintetik foto-shartli virusli ingibitorlari yordamida translokatsiya.[141]

Protein muhandisligi

CEF olimlari tomonidan tuzilmalarning batafsil ma'lumotlaridan foydalanilgan yog 'kislotasi sintazasi (FAS) megakompleksi uchun FAS muhandisiga biosintez ning qisqa zanjirli yog 'kislotalari va poliketidlar, birlashtirilib boshqariladi in vitro va silikonda yondashuv.[142]Ular FAS ning zanjir uzunligini boshqarishni qayta dasturlashdi Saccharomyces cerevisiae qisqa zanjirli yog 'kislotalarini ishlab chiqarishga qodir bo'lgan novvoy xamirturushini yaratish. FAS-larning molekulyar mexanizmlarini o'zgarishsiz qoldirgan va novvoylarning xamirturushida qisqa zanjirli yog 'kislotalari paydo bo'lishiga olib keladigan beshta mutatsiyani aniqlaydigan oqilona va minimal invaziv oqsil muhandislik usuli qo'llanildi.[143] Proteinli fototsiklni yo'naltirilgan usulda boshqarish uchun CEF guruhlari modifikatsiyalash bo'yicha hamkorlik qildilar flavoprotein uning asosiy aminokislotasi bo'lgan dodesin triptofan ularning tarkibiy va elektron ta'siri uchun puxta tanlangan o'rinbosarlar bilan.[144]

CEF tadqiqot yo'nalishi E - makromolekulyar komplekslarni o'rganish usullari

Eng zamonaviy metodologiyalarni ishlab chiqish, shu jumladan elektron paramagnitik rezonans (EPR), vaqt bo'yicha hal qilingan yadro magnit-rezonans spektroskopiyasi (NMR), rivojlangan lyuminestsentsiya mikroskopi, shu qatorda; shu bilan birga optogenetika va optokimyoviy biologiya CEF tadqiqotlarida muhim rol o'ynadi. Klaster shuningdek, yangi ishlanmalarni birlashtirdi elektron mikroskopi va tomografiya kabi super piksellar sonini mikroskopi Riedberg kampusining uslublar portfeliga.

Kriyo-elektron mikroskopi

Kriyo-elektron mikroskopi, 2015 yil tabiat usuli[145] va 2017 yilda Nobel mukofoti berilgan usul[146], bir qancha CEF guruhlari tomonidan keng miqyosda ish bilan ta'minlangan Biofizika MPI shuningdek Gyote universiteti Buchmann Molekulyar Biologiya Instituti.[147][148][149][150][151] Biofizika MPI-ni ishlab chiqishda ishtirok etgan to'g'ridan-to'g'ri elektron detektorlar kutilganidan ham oshib ketdi[152][153]Ushbu detektorlar yordamida tasvirlar, ga qaraganda ancha yuqori kontrast bilan olinishi mumkin CCD kameralar ilgari ishlatilgan va strukturaviy biologiyada ajoyib yutuqlarga olib kelgan. Ushbu yangi texnologiyaga sarmoya kiritib, CEF a'zolari strukturani aniqlashni tezlashtirdilar va makromolekulyar komplekslarning tuzilmalarini echishga muvaffaq bo'ldilar. rentgen kristallografiyasi tadqiqotlar. CEF elektron mikroskopistlarining yana bir yo'nalishi tirik hujayralarning makromolekulyar tashkil topishini aniqlash edi. kriyo-elektron tomografiya. Kriyo-ET - kvazi-tabiiy muhitda buzilmagan hujayralarning molekulyar aniqlikdagi tasvirlarini olishning yagona usuli. Bunday tomogrammalar katta hajmdagi ma'lumotlarni o'z ichiga oladi, chunki ular asosan hujayra proteomining uch o'lchovli xaritasi bo'lib, makromolekulyar o'zaro ta'sirlarning butun tarmog'ini aks ettiradi. Axborot-kon algoritmlar tizimli ma'lumotlardan turli xil texnikalardan foydalanish, alohida makromolekulalarni aniqlash va uyali tomogrammalarga atom rezolyutsiyasi tuzilmalarini hisoblash va shu bilan rezolyutsiya oralig'ini bartaraf etish.[154]

Yorug'lik mikroskopi

Klaster shuningdek, zamonaviy mikroskopning yangi ishlanmalarini qo'llab-quvvatlaydi. Frankfurtdagi tadqiqot texnikasi portfeliga qo'shilgan CEF texnikasi ayniqsa muhimdir yorug'lik varag'i lyuminestsentsiya mikroskopi (LSFM)[155][156]). LSFM-da qo'zg'alish jarayonida optik kesim minimallashtiriladi floroforni oqartirish va fototoksik ta'sir. LSFM biologik namunalari bilan uzoq muddatli uch o'lchovli tasvir yuqori spatiotemporal rezolyutsiyada omon qolganligi sababli, bunday mikroskoplar tanlov vositasiga aylandi rivojlanish biologiyasi. LSFM ta'siri 2015 yilda, jurnal jurnalida tan olingan Tabiat usullari uni "2014 yil uslubi" deb tanladi.[157] CEF olimlari LSFM dan foydalanishdi, masalan, turli xil evolyutsiya bilan bog'liq bo'lmagan hasharotlarning to'liq embrional rivojlanishini tasvirlash va embriondan keyingi o'simlik organlari hujayralari bo'linishi naqshlarining qoidalari va o'z-o'zini tashkil etish xususiyatlarini aniqlash uchun.[158][159][160]Ilg'or nurli mikroskop yordamida ishlab chiqarilgan ma'lumotlarning katta miqdori tasvirlarni avtomatlashtirilgan tahlilini zaruratiga aylantirdi va CEF ma'lumotlarni qayta ishlashni takomillashtirishga va rivojlangan yorug'lik mikroskopi ma'lumotlarini modellashtirishga yordam berdi.[161][162]CEF olimlari tomonidan qo'llaniladigan boshqa yangi nurli mikroskopiya usullariga hujayralardagi biomolekulalarning tarkibiy tuzilishini o'rganish uchun diffraktsiya chegarasidan pastda bitta molekula sezgirligi va fazoviy rezolyutsiyasini ta'minlovchi usullar kiradi. CEF olimlari tomonidan ishlab chiqilgan dasturiy vositalar, masalan, Heidelberg universiteti bilan hamkorlikda ishlab chiqarilgan SuReSim dasturini o'z ichiga oladi, bu erdagi haqiqat modellari bilan ifodalangan o'zboshimchalik bilan uch o'lchovli tuzilmalarning lokalizatsiya ma'lumotlarini simulyatsiya qiladi, bu foydalanuvchilarga o'zgaruvchan eksperimental parametrlarning ko'rish natijalariga qanday ta'sir qilishi mumkinligini muntazam ravishda o'rganishga imkon beradi. .[163] Yangi ishlab chiqilgan texnikani qo'llagan holda CEF olimlari chiziqli rolini aniqlay olishdi hamma joyda sitozolik patogen atrofida palto Salmonella Tifuriy mahalliy NF-kB signalizatsiya platformasi sifatida va bakterial patogenez paytida NF-kB faollashuvida OTULIN funktsiyasi to'g'risida tushunchalar berdi.[164] Yana bir misol - identifikatsiyalash retikulon 3 (RTN3) ning parchalanishi uchun o'ziga xos retseptorlari sifatida ER tubulalar.[165]Konsortsiumning birgalikdagi birgalikdagi ishi jonli hujayralardagi ikkita asosiy muammo va bitta molekulali lokalizatsiya mikroskopini engishga imkon berdi: floroforlarni hujayra membranalari orqali samarali etkazib berish va ultra kichik yorliqlar orqali yuqori zichlikdagi oqsillarni izlash.[166][167] Birgalikda, yangi vositalar hujayra oqsillarini boshqarish va shu bilan birga yuqori molekula asosida bitta molekulali mikroskop bilan o'qish uchun qo'shimcha yo'llarni taqdim etadi.

Spektroskopiya usullari

Keng doirasi spektroskopiya biologik qo'llanilish usullari CEFda mavjud edi va CEF olimlari biomolekulyar NMR va EPRni yanada rivojlantirishda katta yutuqlarga erishdilar. A'zolari Biyomolekulyar magnit-rezonans markazi (BMRZ) suyuqlikning sezgirligini oshirdi- va qattiq holatdagi NMR spektrometr yordamida dinamik yadro polarizatsiyasi (DNP). Tadqiqotchilari bilan birgalikda Rossiya Fanlar akademiyasi, CEF olimlari yuqori quvvatni ishlab chiqdilar girotron DNP uchun manba. Manba 260 gigagertsli chastotada ishlaydi va chiqish quvvati 20 Vtni tashkil qiladi va yarim optik gofrirovka bilan ulanadi to'lqin qo'llanmasi bitta suyuq va bitta qattiq jismga 400 MGts NMR spektrometrga. EPR signalini aniqlaydigan va yuqori quvvatli mikroto'lqinli manbani NMR zondiga ulaydigan mikroto'lqinli karta olimlari bilan hamkorlikda qurilgan. Ukraina Fanlar akademiyasi. Ushbu noyob moslama metallo-dielektrik to'lqinlarni boshqarish tizimiga asoslangan bo'lib, u juda past yo'qotishlarni kafolatlaydi, bu esa asboblar dizayni jihatidan yuqori darajadagi egiluvchanlik bilan birlashtirilgan. CEF olimlari 9. T magnit maydonlarida suvli suyuqliklarda proton NMR signalini 80 baravar ko'payishini namoyish etdilar.[168]Shunday qilib, nazariy bashoratlardan 20 martadan oshib ketdi, makromolekulyar komplekslarga birinchi qo'llanilishlar bir xil darajada muvaffaqiyatli bo'ldi. They also recorded signal enhancements by a factor up to 40 under magic angle sample spinning (MAS) conditions at 100 K with proteorhodopsin re-constituted into lipidli qatlamlar. By integrating DNP-enhanced solid-state NMR spectroscopy with advanced molecular modeling and docking, the mechanism of the subtype selectivity of human kinin G-oqsil bilan bog'langan retseptorlari for their peptide agonists was resolved.[169]DNP-enhanced solid-state NMR spectroscopy enabled CEF scientists to determine the atomic-resolution backbone conformation of an antigenic peptide bound to the human ABC tashuvchisi TAP. Their NMR data also provided unparalleled insights into the nature of the interactions between the side chains of the antigen peptide and TAP. Their findings revealed a structural and chemical basis of substrate selection rules, which define the crucial function of this ABC transporter in human immunitet va sog'liq. This work was the first NMR study of a eukaryotic transporter protein complex and demonstrated the power of solid-state NMR in this field[170] They also demonstrated the power of DNP-enhanced solid-state NMR to bridge the gap between functional and structural data and models.[171]In parallel to the DNP developments, a pulsed electron–electron double resonance (PELDOR) spectrometer with a magnetic field of 6.4 T was constructed. A protein concentration of only 10 pMol is sufficient for a measurement at 40 K. With this instrument, CEF scientists were able to determine the dimerik tuzilishi kovalent bo'lmagan oqsil komplekslari. This method is also applicable to membrane proteins and spin-labelled RNK va DNK molekulalar jonli ravishda.[172] PELDOR spectroscopy proved to be a versatile tool for structural investigations of proteins, even in the cellular environment. In order to investigate for example the structural implications of the asymmetric nucleotide-binding domains and the trans-inhibition mechanism in TAP orthologs, spin-label pairs were introduced via double cysteine mutants at the nucleotide-binding domains and transmembrane domains in TmrAB (a functional homologue of the human antigen translocation complex TAP) and the conformational changes and the equilibrium populations followed using PELDOR spectroscopy.[173] This study defined the mechanistic basis for trans-inhibition, which operates by a reverse transition from the outward-facing state through an occluded conformation. The results uncovered the central role of reversible conformational equilibrium in the function and regulation of an ABC exporter and established a mechanistic framework for future investigations on other medically important transporters with imprinted asymmetry. The study also demonstrated for the first-time the feasibility to resolve equilibrium populations at multiple domains and their interdependence for global conformational changes in a large membrane protein complex.

Ommaviy spektrometriya

Mahalliy mass-spektrometriya has emerged as an important tool in tarkibiy biologiya. Advantages of mass spectrometry compared to other methods like Rentgenologik kristallografiya yoki yadro magnit-rezonansi are for instance its lower limits of detection, its speed and its capability to deal with heterogeneous samples. CEF contributed to the development of laser-induced liquid bead ion desorption mass spectrometry (LILBID), a method developed at Goethe University that is especially suited to the analysis of large membrane protein complexes.[174] A challenge in native mass spectrometry is maintaining the features of the proteins of interest, such as oligomerik state, bound ligandlar, or the conformation of the protein complex, during the transfer from the solution to the gas phase. This is an essential prerequisite to allow conclusions about the solution state protein complex, based on the gas phase measurements. Therefore, soft ionization techniques are required. While standard methods, such as nESI va matritsali lazerli desorbsiya / ionlash (MALDI) reliably deliver valuable results for soluble proteins, they are not universally applicable to the more challenging matrices which are often required for membrane protein complexes. Generally an artificial membrane mimetic environment is required to maintain a membrane protein complex in its native state outside of the cellular environment[175].[176]With LILBID the analitik is transferred into the mass spectrometer in small droplets (30 or 50 µm diameter) of the sample solution produced by a piezo-driven droplet generator and is desorbed from the aqueous solution by irradiation with a mid-IR laser. This results in biomolecular ions with lower, more native-like charge states in comparison to nESI. At ultra-soft desorption conditions, even weakly interacting subunits of large protein complexes remain associated, so that the mass of the whole complex can be determined. At higher laser intensities, the complex dissociates by termoliz and subunit masses are recorded. A broad range of macromolecular complexes from CEF research areas A, C and D, including murakkab I, ATP sintezi, drug transporters with binding proteins, ion kanallari, proteorhodopsins and DNA/RNA complexes, have been analysed using LILBID.[177][178][179][180]

Vaqt bo'yicha hal qilingan spektroskopiya

Femtosekund vaqt bo'yicha hal qilingan spektroskopiya was used by CEF scientists to study molecular dynamics and function. This method enables the observation of extremely fast chemical and biological reactions in real time involving a wide variety of molecules from small organic compounds to complex enzymes. Studies included molecular systems like optical switches, natural and non-natural photosynthetic model systems and membrane protein complexes. Fundamental processes in molecular physical chemistry were investigated, such as photoisomerization, energy and electron transfer and reaction dynamics at surfaces. Modern methods in quantum optics for the generation of appropriately shaped and tunable femtosecond pulses in the visible and infrared spectral range were employed and further developed. Examples of these studies include the investigation and deciphering of the dynamics of photoswitchable or photolabile compounds as basis for the design of photoresponsive biomacromolecules, of the primary reaction dynamics of channelrhodopsin-2 (ChR2) and of the conformational dynamics of antibiotic-binding aptamers: Photochromic spiropyrans are organic molecules that can be used for the triggering of biological reactions.[181][182][183][184][185]

Theoretical biophysics and bioinformatics

Method development in theoretical biophysics plays an increasingly important role in the study of macromolecular complexes and has made essential contributions to many studies in the other research areas of CEF. Bridging between fundamental physics, chemistry and biology, CEF scientists studied biomolecular processes over a broad resolution range, from quantum mechanics to chemical kinetics, from atomistic descriptions of physical processes and chemical reactions in molecular dynamics (MD) simulations to highly coarse-grained models of the non-equilibrium operation of molecular machines and network descriptions of protein interactions. Their goal is to develop detailed and quantitative descriptions of key biomolecular processes, including energy conversion, molecular transport, signal transduction, and enzymatic catalysis. Within CEF, they worked in close collaboration with experimental scientists who employ a wide variety of methods. Their computational and theoretical studies aided in the interpretation of increasingly complex measurements, and guided the design of future experiments.[186][187][188][189][190]The interdisciplinary field of bioinformatics opened new perspectives on molecular processes and cellular function. CEF scientists used custom-tailored code and pipelines for fast and efficient analysis[191]of omics data, with a primary focus on protein-RNA interactions and posttranscriptional regulation.[192] [193][194]They also develops algorithms to solve problems in molecular biology, ranging from atomic protein structure analysis to computational systems biology. Their tools leverage on graph theory, Petri nets and Boolean networks[195] [196]with broad applications within CEF. Their collaborations cover diverse topics from plant metabolomics,[197]to human signal transduction networks[198]and the dissection of the macromolecular complexome.[199][200]

Tashkilot

The CEF Assembly coordinated the research and elected the CEF Speaker and the CEF Board of Directors. The CEF Assembly consisted of the Principal Investigators, Adjunct Investigators, Senior Investigators as well as Associated Members. Speakers of CEF included Werner Müller-Esterl (Nov 2006-Jan 2009), Harald Schwalbe (Feb 2009 - Feb 2013) and Volker Dötsch (March 2013 - October 2019).[201][202]

Nashrlar

CEF scientists published more than 2600 original research publications (incl. 479 research papers in journals with an impact factor of ≥10) during the Cluster's lifetime. To'liq ro'yxatni topish mumkin Bu yerga.

Honours and prizes awarded to CEF scientists

To'liq ro'yxatni topish mumkin Bu yerga.

Tashqi havolalar

Adabiyotlar

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