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Yuvish vositalari hidrofobik membrananing ichki qismiga qanday kiradi?

Yuvish vositalari hidrofobik membrananing ichki qismiga qanday kiradi?


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Ga binoan Molekulyar va hujayrali biologiya (Stiven L. Vulf),

Agar qutb bo'lmagan muhit ta'sir qilsa, membranalar deyarli bir zumda tarqaladi yuvish vositalariBu amfipatik molekulalar bo'lib, ular suv eritmalaridagi membrana lipidlari va oqsillarining hidrofob qismlari atrofida hidrofil qatlam hosil qilishi mumkin.

Bu ahmoqona savol bo'lishi mumkin, lekin ... agar yuvish vositalari membranali osilgan molekulalarning "hidrofob qismlari atrofida palto" yasashi mumkin bo'lsa, ular qandaydir tarzda hidrofob membrananing ichki qismiga kirishi kerak ... to'g'rimi?

Ular membrananing ichki qismiga qanday kiradilar? Ular endositar pufakchalar kabi klasterlar hosil qiladimi? Ular hidrofobik molekula hududlari atrofida hidrofil qatlamlar hosil qilgandan keyin nima bo'ladi?


Bu konsentratsiyaga bog'liq, lekin yuqori konsentratsiyada detarjan molekulalari misel deb ataladi, bu erda hidrofobik "quyruq" ichki qismga, hidrofil "bosh" esa tashqi tomonga yo'naltirilgan. Bu mitselning membrana bilan birlashishi va keyin uni parchalanishiga imkon beradi. Vikipediyadagi ushbu rasm sxematik tarzda ko'rsatilgan:

Bu ko'proq tafsilotlarga ega bo'lgan sirt faol moddalar maqolasidan.


Membran yuvish vositalari bilan tarqatiladi. Ammo yuvish vositalari to'g'ri konsentratsiya va sharoitlarda (tuz, pH va boshqalar) hujayra membranalarini tashkil etuvchi lipidlarga qaraganda kichikroq egrilikka ega mitsellalarni hosil qiladi. Bir oz omad bilan, ular oqsil atrofida kichik hidrofobik mitsel pufakchasini hosil qilishi mumkin.

Bu rasmda membrana oqsilining membrana bilan bog'langan hududlari atrofidagi detarjan misellari tasvirlangan, umid qilamanki, oqsil shaklini buzmasdan!

Siz izohlarda so'raganingiz uchun, bu erda ompF Porin kristallarida detarjan miselining elektron zichligi tasvirlangan - detarjan zichligi ko'k, oqsil qizil. 90-yillarning o'rtalarida, ehtimol bir necha yil oldin, detarjen molekulalarini o'z ichiga olgan juda ko'p simulyatsiyalar mavjud edi. Mishellardagi zanjirlarning harakatchanligi aniq bir model tuzilmasi aniq bo'lmasligini anglatadi, lekin u fotoreaktsiya markazida zaryad o'tkazish yoki import orqali tashish dinamikasi kabi muhim holatlarda erituvchining elektromagnit muhitini tushunishga yordam beradi. oqsilni eksport qilish.


Hidrofobik ta'sir

The hidrofobik ta'sir - qutbsiz moddalarning suvli eritmada to'planish va suv molekulalarini chiqarib tashlash tendentsiyasi. [1] [2] Hidrofobik so'z so'zma-so'z "suvdan qo'rqish" degan ma'noni anglatadi va u suv molekulalari orasidagi vodorod bog'lanishini maksimal darajada oshiradigan va suv bilan qutbsiz bo'lmagan molekulalar orasidagi aloqa maydonini kamaytiradigan suv va qutbsiz moddalarning ajratilishini tavsiflaydi. Termodinamik nuqtai nazardan, hidrofobik ta'sir erigan moddani o'rab turgan suvning erkin energiya o'zgarishidir. [3] Atrofdagi erituvchining erkin energiyaning ijobiy o'zgarishi hidrofobiklikni, manfiy erkin energiyaning o'zgarishi esa hidrofillikni bildiradi.

Hidrofob ta'sir neft va suv aralashmasining uning ikkita komponentiga bo'linishi uchun javobgardir. Shuningdek, u biologiya bilan bog'liq ta'sirlar uchun javobgardir, jumladan: hujayra membranasi va pufakchalarning shakllanishi, oqsillarning katlanishi, polar bo'lmagan lipidli muhitga membrana oqsillarini kiritish va oqsil-kichik molekulalar assotsiatsiyasi. Shunday qilib, hidrofob ta'sir hayot uchun zarurdir. [4] [5] [6] [7] Bu ta'sir kuzatiladigan moddalar hidrofoblar deb ataladi.


Yuvish vositalari hidrofob membrananing ichki qismiga qanday kiradi? - Biologiya

Bu erda biz laboratoriya yuvish vositalari va ularning biotibbiyot tajribalarida qo'llanilishini batafsil ko'rib chiqamiz. Ushbu sharh ionli, ion bo'lmagan va zvitterionli yuvish vositalarini muhokama qilishni, ularning umumiy xususiyatlarini, shuningdek, har bir guruhdagi tez-tez ishlatiladigan yuvish vositalari haqida ma'lumotni o'z ichiga oladi. Nihoyat, biz ba'zi umumiy yuvish vositalari uchun Labome so'rovi natijalarini qisqacha muhokama qilamiz.

Biotibbiyot laboratoriyalarida ishlatiladigan yuvish vositalari yumshoq sirt faol moddalardir (yuzaga ta'sir qiluvchi moddalar), hujayra lizisi (ya'ni, hujayra membranalarining buzilishi) va hujayra ichidagi materiallarni chiqarish uchun ishlatiladi. Ular amfifil molekulalar bo'lib, hidrofilik va hidrofobik hududlarni o'z ichiga oladi. Bu amfifil xususiyat yuvish vositalariga oqsil-oqsil, oqsil-lipid va lipid-lipid assotsiatsiyasini buzish, oqsillarni va boshqa makromolekulalarni denatüratsiya qilish va immunokimyoviy tahlillarda va oqsil kristallanishida nonspesifik bog'lanishni oldini olishga imkon beradi.

Laboratoriya tadqiqotlarida ishlatiladigan yuvish vositalarining ko'p turlari mavjud. Odatda maxsus ilovalar uchun mo'ljallangan yangi amfifil birikmalar ishlab chiqilmoqda (masalan, maltoza-neopentil glikol [1] va glikosil o'rnini bosuvchi dikarboksilatlar [2]). Ushbu maqolada eng ko'p ishlatiladigan laboratoriya yuvish vositalarining xususiyatlari va qo'llanilishi ko'rib chiqiladi.

TuriKimyoviy moddalar
ionlinatriy dodesil sulfat (SDS), deoksixolat, xolat, sarkosil
ion bo'lmaganTriton X-100, DDM, digitonin, 20 yoshli, 80 yoshli
zvitterionikCHAPS
xaotropikkarbamid

Yuvish vositalari amfifil organik birikmalar bo'lib, ular hidrofobik qutbsiz uglevodorod qismi (dum) va gidrofil qutb bosh guruhidan iborat (1A-rasm). Bu molekulyar tuzilish bizning hujayra membranalarini tashkil etuvchi amfifil fosfolipidlarga juda o'xshaydi, faqat fosfolipidlar hidrofil bosh guruhga biriktirilgan juft hidrofob dumlarga ega (1D -rasm). Tegishli konsentratsiyada va haroratda suvda eriganida, amfifil molekulalar o'z-o'zidan tuzilib, tashqi qismidagi gidrofil guruhlarini va ichki qismidagi hidrofob dumlarini suvdan uzoqlashtiradi. Molekulyar farqlari tufayli detarjan molekulalari sharsimon misellarni hosil qiladi (1C -rasm), fosfolipidlar esa ikki qatlamli bo'lish ehtimoli ko'proq (1D -rasm). Molekulyar tuzilmalarning o'xshashligi detarjanning fosfolipid ikki qatlamlariga kirib borishiga va shu bilan hujayra membranalarini buzishiga imkon beradi.

Bundan tashqari, miselning hidrofob yadrosi oqsillarning hidrofob hududlari bilan bog'lanishi mumkin (1B -rasm). Misel tarkibidagi detarjan molekulalari soni yig'ilish raqami deb ataladi, bu membrana oqsillarining eruvchanligini baholash uchun ishlatiladigan muhim parametr [3]. Gidrofob mintaqaning uzunligi hidrofoblik darajasiga to'g'ridan -to'g'ri proportsionaldir va u yuvish vositalari orasida juda doimiy, zaryadlangan guruh esa o'zgaruvchan. Harorat ham, kontsentratsiya ham detarjan fazasini ajratish va eruvchanligining muhim parametrlari hisoblanadi. Ma'lum bir haroratda mitsellar kuzatiladigan minimal detarjen konsentratsiyasi Kritik Misel Konsentratsiyasi (CMC) deb ataladi. CMC dan past bo'lgan har qanday konsentratsiyalarda faqat monomerlar CMC dan yuqori konsentratsiyalarda kuzatiladi, ham mitsellalar, ham monomerlar, suvda erimaydigan boshqa miselyar bo'lmagan fazalar bilan birga mavjud. Xuddi shunday, mitsellar hosil bo'ladigan eng past harorat Critical Micella Temperature (CMT) deb ataladi. CMCga bosh guruhning lipofilligi darajasi ham ta'sir qiladi. Odatda, past lipofil yoki lipofobik xarakter yuqori CMCga olib keladi.

Umumiy yuvish vositalari o'zlarining xususiyatlariga ko'ra uch guruhga bo'linadi: ionli (anion yoki katyonik), ion bo'lmagan va zvitterionik. Quyida men ushbu toifalarning har birida umumiy yuvish vositalarini muhokama qilaman va laboratoriya yuvish vositalarini tanlash va ishlatish haqida muhim ma'lumotlarni taqdim etaman.

Ionik yuvish vositalari hidrofobik zanjir va anionik yoki kationik bo'lishi mumkin bo'lgan zaryadlangan bosh guruhdan iborat. Ular odatda ion bo'lmagan yuvish vositalariga qaraganda yuqori CMC qiymatlariga ega va ular juda qattiq. Zaryadlangan bosh guruhlari tufayli ionli yuvish vositalarini ion almashinadigan xromatografiya yordamida olib bo'lmaydi. Bundan tashqari, ionli yuvish vositalaridan foydalanganda qo'shimcha ehtiyot choralarini ko'rish kerak, chunki ularning ba'zi xususiyatlari o'zgaruvchan ion kuchiga ega buferlarda o'zgarishi mumkin (masalan, NaCl kontsentratsiyasi 0 dan 500 mm gacha ko'tarilganda CMC keskin tushishi mumkin).

Anion SDS ko'pchilik oqsillarni eritishda juda tez-tez ishlatiladigan va samarali sirt faol moddadir. U oqsillar ichidagi va orasidagi kovalent bo'lmagan aloqalarni buzadi, ularni denaturatsiyalaydi va natijada ularning konformatsiyasi va funktsiyalarini yo'qotadi. SDS oqsil zaryadini niqoblab, 1,4: 1 w/w (ikki aminokislotaga taxminan bitta SDS molekulasiga to'g'ri keladi) nisbatidagi oqsil bilan bog'lanadi. Shunday qilib, SDS namunadagi barcha oqsillarga ularning izoelektrik nuqtasidan (pI) qat'i nazar, umumiy manfiy zaryad qo'shadi. Salbiy zaryadlangan SDS molekulalari bilan bog'langanidan so'ng, oqsillarni kattaligiga qarab ajratish mumkin. Bu oqsillarni ajratish va o'rganish uchun SDS poliakrilamid gel elektroforezining (SDS-PAGE) keng qo'llanilishining katta sababidir. Odatda, SDS ishtirokida to'liq hujayrali lizis uchun DNK degradatsiyasini ta'minlash uchun namunani sonikatsiya qilish yoki bir necha marta qirqish kerak (masalan, 19G ignasi orqali). Faol oqsillar zarur bo'lganda yoki oqsil-oqsil o'zaro ta'siri o'rganilayotganda SDSni qo'llash mumkin emas, chunki ularning ikkalasi ham SDS tomonidan buziladi. SDS bilan ishlashda SDS past haroratlarda cho'kishini bilish kerak va bu ta'sir kaliy tuzlari ishtirokida kuchayadi. Bu hodisa ba'zan protein namunasidan SDSni olib tashlash uchun ishlatilishi mumkin [4].

Natriy deoksixolat va natriy xolat - bu safro tuzlari yuvish vositalari. Ularning ikkalasi ham anionik yuvish vositalari. Bu yuvish vositalari ko'pincha membranani buzish va membrana oqsilini olish uchun ishlatiladi, masalan, apelin retseptorlari [5]. Deoksixolat oqsillarni denatüratsiya qiladi, xolat esa denaturatsiya qilmaydigan yuvish vositasidir. Ikkala yuvish vositasi uchun potentsial foyda shundaki, ular oqsillarni miqdoriy va/yoki quyi tahlillarida yordam beradigan dializ yordamida namunalardan olib tashlanishi mumkin.

Sarkosil, shuningdek sarkosil yoki natriy lauroil sarkozinati sifatida ham tanilgan, anion sirt faol moddadir. Hidrofob 14-uglerod zanjiri (lauroil) va gidrofil karboksilat tufayli amfifildir. PKa qiymati 3,6 bo'lgan karboksilat har qanday fiziologik eritmada manfiy zaryadlanadi. Sarkosil lauroilxlorid va sarkozindan natriy gidroksid ishtirokida tayyorlanadi va spirtdan qayta kristallanish yoki mineral kislota bilan kislotalash, erkin kislotani ajratish va erkin kislotani neytrallash yo'li bilan tozalanadi. Sarkosil shuningdek, topikal farmatsevtika mahsulotlarini namlash va kirib borishini yaxshilash uchun ham ishlatilgan. Oziq -ovqat sanoati sohasida sarkosil oziq -ovqat mahsulotlarini qayta ishlash, qadoqlash va tashishda, shuningdek, oziq -ovqat mahsulotlarini saqlash yoki tashishda ishlatiladigan yopishtiruvchi moddalarda foydalanish uchun tasdiqlangan. U shampunlar va tana yuvish vositalari kabi kosmetik formulalarda 3-13% atrofida konsentratsiyalarda keng qo'llaniladi [6]. Sarkosil, shuningdek, kristall modifikatsiyalash, zangga qarshi va korroziyaga qarshi xususiyatlar uchun metall pardozlashda ishlatiladi.

Sarkosil laboratoriya tajribalarida keng qo'llaniladi, masalan, Altsgeymer kasalligida tauni eritishda [7], chunki uning suvda yaxshi erishi, ko'pikning yuqori turg'unligi va oqsillarga kuchli so'rilish qobiliyati bor. Sarkosil hujayralarni o'tkazuvchanligi va oqsillarni ajratish va tozalash usullarida, masalan, Western blot va bilvosita ELISA kabi yuvish vositasi bo'lib xizmat qiladi. Shuningdek, u DNK transkripsiyasining boshlanishini inhibe qilishi mumkin.

Sarkosilning asosiy qo'llanilishi oqsillarni inklyuziya tanalaridan (sitoplazma yoki yadro ichidagi oqsil agregatlari) eritish va qayta tiklash uchundir. Eukaryotik rekombinant oqsillar haddan tashqari ko'payadi Escherichia coli bunday inklyuziv organlarni tuzishga moyildirlar. Sarkosil ko'pincha oqsillarni ajratib olish va ularni tabiiy shaklga qaytarish uchun inklyuziya tanasi pelletini eritish uchun ishlatiladi. Avvalgi ishlar inkubatsiya organlarini karbamid yoki guanidiniy gidroxloridi kabi denaturantlar bilan eritib yuborish va sekin suyultirish yo'li bilan qayta to'ldirish bilan bog'liq edi, ammo erigan oqsillarning ko'pchiligi kuchli yuvish vositalarini olib tashlaganidan keyin yig'ilib cho'kadi. Sarkosil-samarali eriydigan vosita, bu agregatsiyani minimallashtiradi va yuqori oqsil konsentratsiyasida qayta to'kish imkonini beradi (guanidiniy gidroxloridi bilan solishtirganda 10 barobar ko'pdir) [8]. Bir tadqiqot shuni ko'rsatdiki, 95% dan ortiq qo'shma tanadagi sintezlangan oqsillar 10% sarkosil bilan eriydi va oqsillarni boshqa yuvish vositalari (masalan, Triton X-100 va CHAPS) aralashmasi bilan qayta tiklash mumkin [9]. Sarkosil bilan eruvchan ekstraktdagi oqsillar ham afiniteyi tozalashdan oldin bir hafta davomida 4 ° C da saqlanishi mumkin. Shuni ta'kidlash kerakki, sarkosil keyingi xromatografik jarayonga xalaqit beradi va uni eritmadan suyultirish yoki dializ orqali olib tashlash kerak.

Ion bo'lmagan yuvish vositalarida zaryadlanmagan hidrofilik bosh guruhlar mavjud. Ular yumshoq sirt faol moddalar hisoblanadi, chunki ular oqsil-lipid va lipid-lipid assotsiatsiyasini buzadi, lekin odatda oqsil-oqsil o'zaro ta'sirini yo'qotmaydi va odatda oqsillarni denatüratsiya qilmaydi. Shuning uchun ko'plab membrana oqsillari oqsil interaktorlarini saqlab, o'z tabiiy va faol shaklida erishi mumkin. Ammo, barcha oqsillar har xil ion bo'lmagan yuvish vositalari bilan bir xilda harakat qilmagani uchun, siz qiziqqan oqsil (lar) uchun eng yaxshi yuvish vositasini topish uchun sinov va xato kerak bo'lishi mumkin. Shuni ta'kidlash kerakki, ko'p bo'lmagan ionli yuvish vositalari ultrabinafsha (UV) spektrofotometriyaga ta'sir qiladi. Shuning uchun ion bo'lmagan detarjenlar ishtirokida 280 nm da oqsilni aniqlash odatda aniq emas.

Triton oilasining barcha a'zolari: Triton X-100, Triton X-114, Nonidet P-40 (NP-40), Igepal® CA-630 bir-biriga juda o'xshash, ular bir miselga to'g'ri keladigan monomerlarning o'rtacha soni (n) dan farq qiladi. (9,6, 8,0, 9,0 va 9,5 mos ravishda) va ularning polietilen glikol (PEG) asosidagi bosh guruhining o'lchamlari taqsimoti. Ushbu yuvish vositalarining CMC qiymatlari past, shuning uchun ularni dializ yordamida osonlikcha olib bo'lmaydi. Triton X-100, odatda, ion bo'lmagan yuvish vositasi, polioksietilendan olinadi va tarkibida alkilfenil gidrofobik guruh mavjud. Triton X-100 odatda membrana oqsili komplekslarini izolyatsiya qilish uchun ishlatiladi va ko'pchilik uchun tanlangan sirt faol moddasi, masalan, immunopresipitatsiya tajribalari uchun. Triton oilasining boshqa a'zolari past bulutli nuqtalar (mitsellalar to'planib, aniq faza hosil qiladigan harorat) tufayli faza-ajralish yo'li bilan membrana oqsillarini izolyatsiya qilish uchun ishlatiladi. Triton X-100 bulutli nuqtasi 64 daraja, Triton X-114 bulutli nuqtasi 23 daraja. Bu Triton X-114 da membrana oqsilini ajratib olish va eritish imkonini beradi, bu esa namunalarni ko'p oqsillarni denatüratsiya qilishi mumkin bo'lgan issiqroq haroratga olib kelmaydi.

Brij ™ 35 - bu boshqa ion bo'lmagan polioksietilen sirt faol moddasi, u odatda HPLC dasturlarida hujayra lizisi tamponlari yoki tahlil tamponlari yoki sirt faol moddasi sifatida ishlatiladi.

N-dodesil-b-D-maltozid (DDM) glikozid sirt faol moddasi bo'lib, oqsil faolligini saqlab qolish zarur bo'lganda hidrofobik va membrana oqsillarini izolyatsiyalashda tobora ko'proq foydalaniladi. ChP va NP-40 ni o'z ichiga olgan boshqa yuvish vositalariga qaraganda, 2-D elektroforez uchun oqsillarni eritishda samaraliroqdir [10]. Uning lipofil joyidagi glikozanjir, uning yuqori CMC 0,17 mM va mitsellalar interfeysi membrana va hidrofobik oqsillarni eriydi va barqarorligini saqlab qolish uchun ideal suvga o'xshash mikro muhitni yaratadi [11]. Masalan, Winkler MBL va boshqalar NCR1 oqsilini n-dodesil-b-D-maltopiranosid [12] qo'shilishi bilan tozalagan, Li Y va boshqalar LptB2FG va LptB2FGC oqsillari uchun [13]. Steichen JM va boshq., Cryo-EMdan oldin Anatrace DDM eritmasida aralashtirilgan oqsil komplekslari [14].

Boshqa maltozidlar, masalan, beta-desil-maltozid, har xil uzunlikdagi hidrofob alkil zanjirlarga ega. Glyukozid (oktil-glyukozid) oqsillarni tadqiq qilish uchun maltozidli yuvish vositalariga alternativa hisoblanadi [15].

Digitonin, binafsha tulki o'simligidan olingan steroidal glikozid (Digitalis purpurea), hujayra membranalarini eritish uchun ishlatiladi. Bu erda muhokama qilingan boshqa ion bo'lmagan yuvish vositalarida bo'lgani kabi, digitonin tez-tez eriydigan membrana oqsillarini denaturatsiya qilmasdan ishlatiladi. Masalan, B de Laval va boshqalar ATAC-seq protokoli davomida Tn5 transposazasi reaktsiyasi uchun 1% digitoninli hujayralarni lizis qilgan [16]. Chjao Y va boshqalar digitonin bilan postsinaptik zichlikdan sinaptik va ekstrasinaptik AMPA retseptorlarini chiqardilar [17]. Bundan tashqari, digitonin hujayra organellalarini olish uchun ishlatiladi. Digitonin membranalarda xolesterin bilan o'zaro ta'sir qiladi va shuning uchun xolesteringa boy bo'lgan plazma membranasini o'tkazuvchanligini oshirish uchun ishlatilishi mumkin, bunda xolesterin kam bo'lgan organellalar membranalari saqlanib qoladi.

Tween-20 va Tween-80-bu yog'li kislotali efir qismi va uzun polioksietilen zanjiriga ega bo'lgan polisorbat sirt faol moddalar. Ularda CMC juda past, odatda yumshoq sirt faol moddalar, oqsil faolligiga ta'sir qilmaydi va eritishda samarali bo'ladi. Tweens hujayra lizis buferlarining umumiy tarkibiy qismi emas, ammo ular antikorlarning nonspesifik bog'lanishini minimallashtirish va bog'lanmagan qismlarni olib tashlash uchun immunoblotting va Elishayda yuvish vositalari sifatida muntazam ravishda qo'llaniladi va hujayra membranalarini o'tkazish uchun ishlatiladi. Masalan, Yang J va boshqalar HEK293 hujayralarini 0,2% Tween 20 bilan davolashdan so'ng immunostainli hujayra ichidagi FLAG yorlig'i [18].

Tween oilasining yuvish vositalariga oid keng tarqalgan savollardan biri Tween 20 va Tween 80 o'rtasidagi farq, eng ko'p ishlatiladigan ikkita a'zo. Tween 20da laurik kislota, Tween 80da esa oleyk kislota mavjud (3-rasm). 2 -jadvalda ular orasidagi turli jihatlar jamlangan. Ushbu yuvish vositalarini ko'pincha bir-birining o'rnida ishlatish mumkin, ammo ular orasidagi farq ba'zan muhim, masalan in vivo Tween 20 va Tween 80 ning turli darajadagi gemolitik ta'sirining ta'siri bo'lishi mumkin bo'lgan tadqiqotlar [19]. Masalan, Greenwood DJ va boshqalar o'sdi Mycobacterium tuberculosis 0,05% Tween 80 bilan to'ldirilgan muhitda [20]. Ouadah Y va boshqalar sichqonlarga Notch signalizatsiyasini inhibe qilish uchun 0,1% v/v Tween 80 bilan dibenzazepin eritmasini yuborishdi [21].

Sinonimlar Kimyoviy formula Molekulyar Og'irligi Zichlik (g/ml) Tashqi ko'rinishi Ilovalar
20 o'rtasidapolisorbat 20, polioksietilen sorbitan monolaurat, PEG (20) sorbitan monolauratC 58 H 114 O 26 12281.1Shaffof, sariqdan sariq-yashil ranggacha bo'lgan yopishqoq suyuqlikilovalarning keng doirasi: Elishay uchun PBS yoki TBS yuvish tamponlarida blokirovka qiluvchi vosita sifatida, sut emizuvchilar hujayralarini lizinglash uchun Western blot va boshqa immunoassay usullari va membrana oqsillari uchun erituvchi vosita sifatida.
80 o'rtasidapolisorbat 80, polioksietilen sorbitan monooleat, PEG (80) sorbitan monooleatC 64 H 124 O 26 13101.06-1.09kehribar rangli yopishqoq suyuqlikvaktsina preparatlarida ishlatiladigan ba'zi mikobakteriyalarning fenotipini aniqlash uchun testlarda qo'llaniladigan oqsillar uchun stabillashtiruvchi vosita sifatida [22]

Ion bo'lmagan yuvish vositalari odatda nisbatan yumshoq bo'lsa-da, ko'plab oqsillar bu yuvish vositalarining mavjudligida denatüratsiyalanadi yoki yig'iladi. Ushbu muammoni hal qilish uchun yangi ion bo'lmagan gliko-litoxolat amfifillari (GLC-1, GLC-2 va GLC-3) va gliko-diosgenin amfifillari (GDN) ishlab chiqildi [23]. GDN oqsilning qanday ishlashini yaxshiroq tushunish uchun xamirturush mitoxondriyal dimerik ATP sintazini olish uchun ishlatilgan [24]. Pluronik F-68 odatda suvni kesish kuchini kamaytirish uchun 0,1% suspenziya hujayrali kulturasida ishlatiladi [25].

Zvitterionik yuvish vositalarining bosh guruhlari hidrofil bo'lib, ular musbat va manfiy zaryadlarni teng miqdorda o'z ichiga oladi, natijada aniq zaryad nol bo'ladi. Ular ion bo'lmagan yuvish vositalariga qaraganda qattiqroq sirt faol moddalardir. Oddiy zvitterionik yuvish vositasi 3-[(3-xolamidopropil)dimetilamonio]-1-propansulfonat bo'lib, CHAPS nomi bilan mashhur. CHAPS yuqori CMC (xona haroratida 6 mM) dializ orqali samarali olib tashlash imkonini beradi. Izoelektrik fokuslash va 2D elektroforez uchun 2-4% konsentratsiyalarda namuna tayyorlashda juda keng tarqalgan. CHAPSO ning CHAPSdan farqi shundaki, u ko'proq qutbli bosh guruhni o'z ichiga oladi, bu esa uni hidrofob molekulalarni eritishga qodir qiladi. Shunday qilib, CHAPSO asosan ajralmas membrana oqsillarini eritish uchun ishlatiladi.

Xaotrop moddalar sirt faol moddalarga o'xshash moddalar bo'lib, ular kovalent bo'lmagan o'zaro ta'sirlarni (vodorod aloqalari, dipol-dipol o'zaro ta'sirlari, hidrofobik o'zaro ta'sirlar) buzadi, bu holda oqsil denatüratsiyasini osonlashtiradi, bu holda odatda qaytariladi. Karbamid-bu 2D-gel elektroforezi va proteomik ish jarayonida tayyorgarlik uchun oqsillarni eritma-fermentativ hazm qilish kabi dasturlarda yakka o'zi yoki tiyoamid yoki boshqa yuvish vositalari bilan birgalikda ishlatiladigan umumiy xaotrop vosita. Karbamiddan foydalanganda, namunani 37 ° C dan yuqori qizdirmaslikka alohida e'tibor berish kerak, chunki bu oqsillarning karbamillanishiga olib keladi [26].

Membranada oqsilning eruvchanligi uchun odatda yuqori CMC bo'lgan yuvish vositasini tanlash kerak, shuningdek, buferning hajmi va kontsentratsiyasi ham hal qiluvchi ahamiyatga ega, chunki namunadagi barcha membrana oqsillarini eritish uchun etarli miqdorda yuvish vositasi bo'lishi kerak. Ko'pgina hollarda, membrana oqsillarini eritish uchun etarli misel konsentratsiyasini ta'minlash uchun detarjan konsentratsiyasi CMC darajasida yaxshi bo'lishi kerak (kamida 2x CMC). Linke [3] ga koʻra, membrananing lipid muhitini yetarlicha taqlid qilish uchun membrana oqsili molekulasiga kamida bitta mitsel kerak (1B, D-rasm).

Proteinlarni yanada tozalash uchun fazani ajratish mumkin. Bu buferdagi harorat va tuzlar va yuvish vositalarining kontsentratsiyasini sozlashni talab qiladi, shunda detarjan misellari yig'ilib suvli qatlamdan ajralib chiqadi. Bunday holda, mitsellalar bilan o'ralgan membrana oqsillari yuvish vositasi bilan birlashadi. Detarjan eritmasi ikki fazaga ajraladigan haroratga, bulutli nuqtaga buferdagi glitserin yoki tuzlar ta'sir qiladi (masalan, Triton X-114 ning bulutlanish nuqtasi 23°C, lekin 20% glitserin ishtirokida, bulut nuqtasi 4 ° C gacha pasayadi). Bu juda muhim, chunki oqsilning barqarorligiga yuqori harorat ta'sir qiladi.

Yaxshi yuvish vositasi hujayralarni ajratishi, oqsillarni eritishi va sizning quyi oqimdagi ilovalaringizga mos bo'lishi kerak. Bundan tashqari, tabiiy yoki denatüratsiyalangan shaklda erigan oqsilni hisobga olish kerak. Barcha dasturlar uchun ideal yuvish vositasi yo'q va hatto bir xil dasturda ham natija turlicha bo'ladi (3 -jadval). Shuning uchun, variantlar ko'rib chiqilgandan so'ng, eng yaxshi yuvish vositasini topish uchun ko'pincha sinov va xato kerak bo'ladi va yuvish vositalarining aralashmasi optimal bo'lishi mumkin. Bundan tashqari, detarjan ishchi eritmasini yangi tayyorlash odatda gidroliz va oksidlanishdan saqlanish uchun eng yaxshi amaliyotdir.

Yuvish vositasiMVt (Da) monomerMVt (Da) mitselCMC (mm) 25 o CAgregatsiya raqamiBulut nuqtasi (o C)O'rtacha Miselyar og'irlikKuchDializ mumkinIlovalar
SDS28918,0007-1062>10018,000QattiqHaHujayra lizisi, elektroforez, JB, duragaylash
Triton X-10062590,0000.2-0.9100-1556580,000YumshoqYo'qImmunologik tahlillar, IP, membranalarda erishi
BOBLAR6156,150610>1006,150YumshoqHaIEF, IP
NP-4068090,0000.059 45-50 YengilYo'qXEF
n-dodesil-b-D-maltozid511 0.1598 50,000 Proteinning kristallanishi
Tween-201228 0.06 76 YumshoqYo'qWB, Elishay, ferment immunoassays
Digitonin122970,000<0.560 70,000YumshoqYo'qMembrananing erishi

Pastki oqim ilovalari ko'pincha detarjan konsentratsiyasini kamaytirishni yoki butunlay olib tashlashni talab qiladi. Bunday maqsadlarda, agar mitsellar o'lchami qiziqishdagi oqsildan sezilarli darajada farq qilsa yoki mitsellar dializ trubkasidan o'tish uchun etarlicha kichik bo'lsa (ya'ni, yuqori CMC) bo'lsa, o'lchamni istisno qilish xromatografiyasi yoki dializdan foydalanish mumkin [3]. Boshqa usullar detarjan bilan bog'laydigan qutbsiz boncuklar yoki qatronlar, siklodekstrin inklyuziya aralashmalari [27], ion almashinadigan kromatografiya yoki oqsillarni cho'ktirishdan foydalanishni qo'llaydi. Shu bilan birga, detarjan olib tashlanganidan keyin ishlatiladigan tampon protein cho'kishi yoki agregatsiyasini oldini olish uchun ehtiyotkorlik bilan tanlanishi kerak.

Labome yuvish vositalarini qo'llash bo'yicha adabiyotlarni o'rganadi. Quyidagi jadvalda asosiy etkazib beruvchilar va kir yuvish vositalarining aksariyati MilliporeSigma tomonidan etkazib beriladigan mahsulotlar soni ko'rsatilgan.

yuvish vositasi yetkazib beruvchilar
Triton X-100Termo Fisher [28, 29], elektron mikroskopiya fanlari [30], Amresko, JT Beyker
20 yoshgachaBio-Rad [31], MilliporeSigma [29], Termo Fisher
SDSAmresco, Bio-Rad, Q.BIOgene, MilliporeSigma
NP-40Roche, MilliporeSigma [32]
CHAPSMilliporeSigma, JT Beyker
digitoninMilliporeSigma, Vako
DDMGeneron [33], Anatrace [15]

Termo Fisher Pirs Triton X-100, masalan, BP151 [29] yoki 85111 [28], immunohistokimyo [29] va immunotsitokimyo [28] uchun hujayra va to'qimalar namunalarini lizlash uchun ishlatilgan. MilliporeSigma Triton X-100 hujayralarni lizlashda [34] yoki immunotsitokimyoda [35] hujayralarni o'tkazuvchanlikda, immunohistokimyoda [36, 37] va proteinaz K himoyasi tahlilida [38] blokirovkalash buferida ishlatilgan.

Tween-20 odatda TBS-Tween (TBS-T) yoki PBS-Tween (PBT-T) kabi buferlarni yuvishda har xil immunoassaylarda ishlatiladi. MilliporeSigma Tween-20, masalan, P1379 [29] yuvish blotlarida [29], IHC tajribalarida (P1379) [39], immunopresipitatsiyada [40] va mikrosuyuqlik massivi multipleks PCRda [41] va boshqalarda foydalanilgan. 42]. MilliporeSigma Tween-80, erlotinibni (kimyoterapiya dori) [43] eritishda va o'sish uchun qo'shimcha sifatida ishlatilgan. M. sil kasalligi shtammlar [44].

Xromatin preparatlarida Lonza SDS (katalog raqami 51213) ishlatilgan [39]. Amresco SDS SDS-PAGE [45] da ishlatilgan. Bio-Rad natriy dodesil sulfat radioimmunopresipitatsiya tahlili tamponini tayyorlash uchun ishlatilgan [46]. MilliporeSigma-Aldrich SDS in vitro oktanoillanish tahlillari, Laemmli namuna buferi, 2D-DIGE tajribalari [47] uchun buferlarni tayyorlash uchun ishlatilgan.

Roche NP-40 hujayra lizisida ishlatilgan [48, 49]. MilliporeSigma NP-40 radioimmunopresipitatsiya tahlili buferini [46], hujayra lizisini/gomogenizatsiya buferini [50, 51] va immunoprecipitatsiya tahlilini RIPA buferini [52] tayyorlash uchun ishlatilgan.

MilliporeSigma CHAPS protein kristallanishi uchun tamponlarda ishlatilgan [53]. JT Baker CHAPS insonning ASF1 oqsili bilan virusli o'zaro ta'sirini o'rganish uchun hujayralarni lizinglash uchun ishlatilgan [54].

MilliporeSigma immunotsitokimyoviy eksperimentda PI4P [55] ni o'rganish uchun ishlatilgan va proteinaz K himoyasi tahlillarini [38] o'tkazish va RNKni ajratish uchun ishlatilgan [56]. Vako digitonin hujayralarni parchalash [57] va immunopresipitatsiya tajribalarini [58] bajarish uchun ishlatilgan.

Y Li va boshqalar Generondan [33] dodesilmaltozid / DDM bilan GPCR oqsilini eritdilar. Proteinni tozalash uchun Anatrace n-desil-beta-D-maltopiranozid ishlatilgan [59, 60], n-dodesil-beta-D-maltozid [61] va n-undesil-beta-D-maltozid [62, 63]. . Glikon beta-dodesil-maltozid va beta-desil-maltozid oqsillarni tozalashda ham ishlatilgan [64]. Anatrace n-oktil-beta-glyukozid AQP4 oqsillarini eritishda ishlatilgan [15].

Silva MC va boshqalar Brij-35 ni bio-qatlam interferometriyasi biosensorini tahlil qilish uchun yuvish vositasi sifatida ishlatgan [65]. Proteinni tozalash uchun Affymetrix oktil glyukoza neopentil glikol (OGNPG) 1% [66] va MilliporeSigma xolesteril hemisuksinat 0,1% yoki 0,05% (w/v) [11, 67] ishlatilgan. Xromatin bilan bog'liq tahlillar uchun MilliporeSigma-Aldrich natriy deoksixolat (katalog raqami D6750) va Igepal (katalog raqami I8896) va TEKnova N-lauroilsarkosin (katalog raqami S3379) ishlatilgan [39].


Hujayra lizisi va oqsil olish uchun yuvish vositalari

Yuvish vositalari amfipatik molekulalardir, ya'ni ular alifatik yoki aromatik xarakterga ega bo'lgan qutbsiz "quyruq" ni va qutbli "boshni" o'z ichiga oladi. Qutb bosh guruhining ion xarakteri yuvish vositalarini keng tasniflash uchun asos bo'lib xizmat qiladi, ular ionli (zaryadlangan, anion yoki katyonik), noionik (zaryadlanmagan) yoki zvitterionik (musbat va manfiy zaryadlangan guruhlarga ega, ammo sof zaryadi nolga teng) bo'lishi mumkin. ).

Eritmadagi yuvish vositalari

Biologik membranalarning tarkibiy qismlari singari, yuvish vositalari ham qutbsiz quyruq guruhlari natijasida hidrofobik bog'lovchi xususiyatlarga ega. Shunga qaramay, yuvish vositalarining o'zi suvda eriydi. Binobarin, detarjan molekulalari suvda erimaydigan, hidrofob birikmalarning suvli muhitga tarqalishiga (aralashib ketishiga), shu jumladan membrana oqsillarini ajratib olish va eritishga imkon beradi.

Suvli eritmada past konsentratsiyali yuvish vositalari havo-suyuqlik interfeysida mono qatlam hosil qiladi. Yuqori konsentratsiyada detarjen monomerlari birlashib, misellar deb ataladi. Misel - bu termodinamik jihatdan barqaror kolloid detarjenli monomerlar agregati bo'lib, ularda qutbsiz uchlari suvga tegmaslik uchun ichkariga joylashtiriladi va qutbli uchlari suv bilan aloqa qilganda tashqariga yo'naltiriladi.

Detarjan miselining ideal tuzilishi.

Har bir miselga to'g'ri keladigan deterjan monomerlari soni (yig'ilish raqami) va yuqorida misellar hosil bo'ladigan detarjan kontsentratsiyasining diapazoni (kritik misel konsentratsiyasi, CMC deb ataladi) har bir detarjanga xos xususiyatlardir (jadvalga qarang). Kritik misel harorati (CMT) - bu misellar hosil bo'lishi mumkin bo'lgan eng past harorat. CMT bulut nuqtasi deb ataladigan narsaga to'g'ri keladi, chunki detarjan mitsellari CMT dan past haroratlarda kristalli suspenziyalar hosil qiladi va CMT dan yuqori haroratlarda yana tiniq bo'ladi.

Yuvish vositalarining xususiyatlariga konsentratsiya, harorat, bufer pH va ion kuchi va turli qo'shimchalarning mavjudligi kabi eksperimental sharoitlar ta'sir qiladi. Masalan, ba'zi noonik yuvish vositalarining CMC harorat ortishi bilan kamayadi, ionli yuvish vositalarining CMC esa zaryadlangan bosh guruhlar orasidagi elektrostatik itarilishning kamayishi natijasida qarshi ion qo'shilishi bilan kamayadi. Boshqa hollarda, karbamid kabi qo'shimchalar suv tarkibini samarali ravishda buzadi va CMC detarjanining pasayishiga olib keladi. Umuman olganda, yig'ilish sonining keskin o'sishi ion kuchining oshishi bilan sodir bo'ladi.

Yuvish vositalari oqsil tuzilishiga nisbatan denatürasyonlu yoki denatüre bo'lmagan bo'lishi mumkin. Denaturatsiya qiluvchi yuvish vositalari natriy dodesil sulfat (SDS) kabi anionik yoki etil trimetil ammoniy bromid kabi katyonik bo'lishi mumkin. Ushbu yuvish vositalari oqsil-oqsil o'zaro ta'sirini buzish orqali membranalarni butunlay buzadi va oqsillarni denatüratsiya qiladi. Denaturatsiya qilmaydigan yuvish vositalarini Triton X-100 kabi noonik yuvish vositalariga, xolat kabi safro tuzlariga va CHAPS kabi zvitterionik yuvish vositalariga bo'lish mumkin.

Umumiy yuvish vositalarining xususiyatlari.

Yuvish vositasiTuriAgg.#‡MVt
mono
(mitsel)
CMC
mm
(%v/v)
Bulut
nuqta
° C
Dializ mumkin
Thermo Scientific Triton X-100Noionik140647 (90K)0.24 (0.0155)64Yo'q
Thermo Scientific Triton X-114Noionik537 ( – )0.21 (0.0113)23Yo'q
NP-40Noionik149617 (90K)0.29 (0.0179)80Yo'q
Termo ilmiy Brij-35Noionik401225 (49K)0.09 (0.0110)& gt100Yo'q
Termo ilmiy Brij-58Ion bo'lmagan701120 (82K)0.08 (0.0086)>100Yo'q
Thermo Scientific Tween 20Ion bo'lmagan1228 ( – )0.06 (0.0074)95Yo'q
Thermo Scientific Tween 80Noionik601310 (76K)0.01 (0.0016)Yo'q
Oktil glyukozidIon bo'lmagan27292 (8K)23-24 (

‡Agg.# = Aggregatsiya raqami, bu mitseldagi molekulalar soni.

Tozalangan detarjen eritmalari

Yuvish vositalari bir nechta tijorat manbalaridan olinsa va ko'plab tadqiqot laboratoriyalarida muntazam qo'llanilsa -da, detarjan tozaligi va barqarorligining ahamiyati keng baholanmaydi. Yuvish vositalarida ko'pincha ularni ishlab chiqarishda iz qoldiqlari mavjud. Ushbu aralashmalarning ba'zilari, ayniqsa noonik yuvish vositalarida mavjud bo'lgan peroksidlar oqsil faolligini yo'q qiladi. Bundan tashqari, kir yuvish vositalarining bir qancha turlari havo yoki ultrabinafsha nurlar ta'sirida tezda oksidlanib, eriydigan moddalar sifatini yo'qotadi. Biz shaffof shisha ampulalarda azot gazi ostida qadoqlangan bir nechta yuqori toza, past peroksid o'z ichiga olgan yuvish vositalarini taklif qilamiz. Ushbu Thermo Scientific Surfact-Amps detarjan yechimlari barcha yuvish vositalarini qo'llash uchun misli ko'rilmagan qulaylik, sifat va mustahkamlikni ta'minlaydi. Namuna oluvchi to'plamga 10 xil tozalangan yuvish vositalari kiradi (7 tasi Surfact-Amps formatida va uchtasi qattiq shaklda).

Batafsil ma'lumot

Mahsulotlarni tanlang

Hujayra membranalarining tuzilishi

A major factor determining the behavior and interaction of molecules in biological samples is their hydrophilicity or hydrophobicity. Most proteins and other molecules with charged or polar functional groups are soluble (or miscible) in water because they participate in the highly ordered, hydrogen-bonded intermolecular structure of water. Some other proteins (or at least parts of proteins), as well as fats and lipids, lack polar or charged functional groups consequently, they are excluded from the ordered interaction of water with other polar molecules and tend to associate together in structures having minimal surface area contact with the polar environment. This association of nonpolar molecules in aqueous solutions is commonly called hydrophobic attraction, although it is more accurately understood as exclusion from the hydrophilic environment.

The formation and stability of biological membranes results in large measure from the hydrophobic attraction of phospholipids, which form bilayer sheets having hydrophobic lipid "tails" oriented within the sheet thickness and polar "head" groups oriented to the outer and inner aqueous environments. Membrane proteins completely span the membrane thickness or are embedded at one side of the membrane in accord with their structure of hydrophobic and hydrophilic amino acid side chains and other functional groups.

Membrane disruption, protein binding and solubilization

Generally, moderate concentrations of mild (i.e., nonionic) detergents compromise the integrity of cell membranes, thereby facilitating lysis of cells and extraction of soluble protein, often in native form. Using certain buffer conditions, various detergents effectively penetrate between the membrane bilayers at concentrations sufficient to form mixed micelles with isolated phospholipids and membrane proteins.

Detergent-based cell lysis. Both denaturing and non-denaturing cell lysis reagents may be used for protein extraction procedures.

Denaturing detergents such as SDS bind to both membrane (hydrophobic) and non-membrane (water-soluble, hydrophilic) proteins at concentrations below the CMC (i.e., as monomers). The reaction is equilibrium driven until saturated. Therefore, the free concentration of monomers determines the detergent concentration. SDS binding is cooperative (i.e., the binding of one molecule of SDS increases the probability that another molecule of SDS will bind to that protein) and alters most proteins into rigid rods whose length is proportional to molecular weight.

Non-denaturing detergents such as Triton X-100 have rigid and bulky nonpolar heads that do not penetrate into water-soluble proteins consequently, they generally do not disrupt native interactions and structures of water-soluble proteins and do not have cooperative binding properties. The main effect of non-denaturing detergents is to associate with hydrophobic parts of membrane proteins, thereby conferring miscibility to them.

At concentrations below the CMC, detergent monomers bind to water-soluble proteins. Above the CMC, binding of detergent to proteins competes with the self-association of detergent molecules into micelles. Consequently, there is effectively no increase in protein-bound detergent monomers with increasing detergent concentration beyond the CMC.

Detergent monomers solubilize membrane proteins by partitioning into the membrane bilayer. With increasing amounts of detergents, membranes undergo various stages of solubilization. The initial stage is lysis or rupture of the membrane. At detergent:membrane lipid molar ratios of 0.1:1 through 1:1, the lipid bilayer usually remains intact but selective extraction of some membrane proteins occurs. Increasing the ratio to 2:1, solubilization of the membrane occurs, resulting in mixed micelles. These include phospholipid–detergent micelles, detergent–protein micelles, and lipid–detergent–protein micelles. At a ratio of 10:1, all native membrane lipid:protein interactions are effectively exchanged for detergent:protein interactions.

The amount of detergent needed for optimal protein extraction depends on the CMC, aggregation number, temperature and nature of the membrane and the detergent. The solubilization buffer should contain sufficient detergent to provide greater than 1 micelle per membrane protein molecule to help ensure that individual protein molecules are isolated in separate micelles.

Detergents used for cell lysis. Major characteristics of denaturing and non-denaturing detergents used for protein extraction.


How Detergents Work

Neither detergents nor soaps accomplish anything except binding to the soil until some mechanical energy or agitation is added into the equation. Swishing the soapy water around allows the soap or detergent to pull the grime away from clothes or dishes and into the larger pool of rinse water. Rinsing washes the detergent and soil away.

Warm or hot water melts fats and oils so that it is easier for the soap or detergent to dissolve the soil and pull it away into the rinse water. Detergents are similar to soap, but they are less likely to form films (soap scum) and are not as affected by the presence of minerals in the water (hard water).


Content Background: How Does an Anabolic Steroid Reach its Target?

Once in the bloodstream, the anabolic steroid travels to all tissues in the body, where it enters the cells to reach its target. In order to get into a muscle cell for example, the steroid must leave the capillary and then enter the muscle cell. This means that the steroid must cross two different types of membranes, the capillary membrane and the muscle cell membrane. To cross the capillary membrane, there are numerous pores or fenestra 1 , which allow small molecules to squeeze through (Figure 3 and see Module 1). However the muscle cell membrane (like most cells in the body) does not have these small pores and therefore the steroid can only cross the membrane by diffusing across or by transport via a carrier protein. Steroids cross the cell membrane by passive diffusion 2 , which occurs in the direction of the concentration gradient – this does not require energy. Passive diffusion depends on the physiochemical characteristics of the membrane and the drug 3 . The muscle cell membrane, like all cell membranes in the body, is a lipid bilayer (Figure 4). It consists of lipids arranged with their polar 4 head groups facing the outside and inside of the cell. The chains of fatty acids face each other, forming the hydrophobic 5 (water-fearing) or non-polar 6 interior. Because anabolic steroids 7 are very lipophilic 8 (lipid-loving), they diffuse easily into the hydrophobic membrane interior. As they concentrate within the hydrophobic membrane interior, a new driving force is generated, pushing the steroid into the cytoplasmic side of the cell membrane. Once the anabolic steroid diffuses into the cytoplasm of the cell, it binds to the androgen receptor 9 (Figure 5). [Receptors for other steroids are found in the nucleus instead of the cytoplasm.] This complex of steroid and protein then crosses the nuclear membrane to enter the nucleus of the cell, where it exerts its effects. In this case, passive diffusion can’t occur because the protein is too large and not lipophilic. Instead, the steroid-receptor complex moves through small pores in the nuclear membrane to enter the nucleus. Although scientists are still elucidating exactly how this occurs, it is possible that the complex interacts with transport proteins that line the nuclear pores. This is an example of facilitated diffusion 10 , which occurs in the direction of the concentration gradient. Therefore, no energy is required. This is unlike active transport 11 , which occurs against the concentration gradient, and requires energy.

Definitions:
1 small spaces or pores within endothelial cells that form the capillary membrane. These pores allow charged drugs or larger drugs to pass through the capillaries.
2 the movement of a solute in its uncharged form to cross a membrane along a concentration gradient. No energy is required.
3 a substance that affects the structure or function of a cell or organism.
4 a chemical property of a substance that indicates an uneven distribution of charge within the molecule. A polar substance or drug mixes well with water but not with organic solvents and lipids. Polar or charged compounds do not cross cell membranes (lipid) very easily.
5 “water-fearing” a compound that is soluble in fat but not water. This is typical of compounds with chains of C atoms.
6 a chemical property of a substance that indicates an even distribution of charge within the molecule. A non-polar or non-charged compound mixes well with organic solvents and lipids but not with water.
7 synthetic versions of testosterone designed to promote muscle growth without producing androgenic effects. The better term is anabolic-androgenic steroid.
8 high lipid solubility. Lipophilic compounds dissolve readily in oil or organic solvent. They exist in an uncharged or non-polar form and cross biological membranes very easily.
9 a protein to which hormones, neurotransmitters and drugs bind. Ular odatda hujayra membranalarida joylashgan va bir marta bog'langan funktsiyani keltirib chiqaradi.
10 the movement of molecules across a membrane with the concentration gradient. No energy is required, but transport proteins can become saturated, limiting the diffusion process.
11 the movement of molecules against the concentration gradient with the help of a transport protein. This transport requires energy in the form of ATP.

3 -rasm A capillary is composed of endothelial cells that connect together loosely. Small pores or fenestrae are also present, allowing solutes to move in and out of the capillaries.

4-rasm Schematic view of cell membrane. Lipids are arranged with polar head-groups facing the outside and inside of the cell, while the fatty acid chains form the non-polar (hydrophobic) membrane interior.

5 -rasm Testosterone (or anabolic-androgenic steroids) binds to the androgen receptor in the cytoplasm and the complex moves into the nucleus where it interacts with DNA to initiate protein synthesis.


Detergents

Detergents are critical tools for the study of membrane proteins. They are vital for the isolation and purification of the proteins and are used in the primary solubilization step of reconstitution. They are invaluable in membrane protein recrystallization.

So What are detergents? Detergents are soluble amphiphilic molecules consisting of a polar head group and hydrophobic chain (or tail) and exhibit unique properties in aqueous solutions in which they spontaneously form spherical micellar structures. Membrane proteins are frequently soluble in micelles formed by amphiphilic detergents. Detergents solubilize membrane proteins by creating a mock lipid bilayer environment normally inhabited by the protein.

3 Major Detergent Classifications

    • Ionic Detergents: These have a polar head that can be either anionic or cationic and a hydrophobic chain or tail with a steroidal backbone. They are very efficient at solubilizing proteins, but almost always cause denaturation of the protein to some extent. An example of an ionic detergent is Sodium Dodecyl Sulfate (SDS).

      • Non-Ionic Detergents: These detergents have an uncharged hydrophilic head of either Polyoxyethylene or glycosidic group. It is a relatively mild detergent that solubilizes proteins by breaking the lipid-lipid interactions or lipid-protein interactions. Ionic detergents do not break the protein-protein interactions thereby, the solubilized protein is structurally intact in its biological form. Ionic detergents are effective in isolating active membrane proteins. Examples of Non-Ionic detergents are - n-Octyl-β-D-glucopyranoside (OG) and n-dodecyl-β-D-maltoside(DDM).
      • Zwitterionic Detergents: The polar head groups of zwitterionic detergents have a neutral charge. They have both ionic and non-ionic properties. The strength of action of zwitterionic detergents is intermediate between both ionic and non-ionic detergents.

      Zwitterionic Detergents for Membrane Proteins

      Zwitterionic detergents are efficient at breaking protein-protein interactions and are less harsh than ionic detergents. While zwitterionic detergents break the protein bonds, they are still successful at maintaining the native state and charge of individual proteins.
      Due to their versatility, Zwitterionic detergents are useful in a variety of applications such as:

      • Xromatografiya
      • Different types of electrophoresis, including 2D gel electrophoresis
      • Mass spectrometry
      • Solubilization of organelles and inclusion bodies.

      Examples of zwitterionic detergents include CHAPS and CHAPSO.

      Synthetic zwitterionic detergents are known as sulfobetaines. This group of substances retain their zwitterionic characteristics over a wide pH range.


      YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis

      Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopN W279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.

      Kalit so'zlar: bacterial pathogenesis molecular modeling mutagenesis protein secretion protein-protein interaction regulation.

      Raqamlar

      32 kDa) polypeptide, while the double asterisk ( ** ) reveals the naturally produced and secreted

      42 kDa YopN-TyeA hybrid. The arrows (←) indicate a non-specific protein band recognized by the anti-YopN antiserum and the anti-YopD antiserum. The band appearing just above the nonspecific band in the ΔtyeA strain likely represents a frameshifting event that causes full-length YopN to be fused with the TyeAΔ19−59 deletion remnant resulting in a hybrid product that has a predicted molecular weight of

      38 kDa. Strains: Parent (YopNmahalliy), YPIII/pIB102 ΔyscU, lcrQ double mutant, YPIII/pIB75-26 ΔyopN null mutant, YPIII/pIB82 ΔtyeA null mutant, YPIII/pIB801a ΔyopN, tyeA double mutant, YPIII/pIB8201a Mutant 1–YopN288(scramble)293, YPIII/pIB8213 Mutant 2–YopN288STOP, YPIII/pIB8212 Mutant 3–YopN279(F+1), 287(F−1), YPIII/pIB8208 Mutant 4–YopN279(F+1), 287STOP, YPIII/pIB8207 Mutant 5–YopN279STOP, YPIII/pIB8209. The theoretical molecular masses predicted from amino acid sequence are given in parentheses.


      How do detergents get in hydrophobic membrane interior? - Biologiya

      Detergents are invaluable tools for studying membrane proteins. However, these deceptively simple, amphipathic molecules exhibit complex behavior when they self-associate and interact with other molecules. The phase behavior and assembled structures of detergents are markedly influenced not only by their unique chemical and physical properties but also by concentration, ionic conditions, and the presence of other lipids and proteins. In this minireview, we discuss the various aggregate forms detergents assume and some misconceptions about their structure. The distinction between detergents and the membrane lipids that they may (or may not) replace is emphasized in the most recent high resolution structures of membrane proteins. Detergents are clearly friends and foes, but with the knowledge of how they work, we can use the increasing variety of detergents to our advantage.

      Published, JBC Papers in Press, June 29, 2001, DOI 10.1074/jbc.R100031200

      This minireview will be reprinted in the 2001 Minireview Compendium, which will be available in December, 2001. This is the third article of four in the “Membrane Protein Structural Biology Minireview Series.” Some of the work discussed in this minireview was supported in part by National Institutes of Health Grants P01 GM57323 (to R. M. G. and S. F. M.) and HL56773 (to R. M. G.).

      This minireview is dedicated to Drs. Jacqueline A. Reynolds and the late Martin Zulauf who gave one of us (R. M. G.) invaluable insights into the behavior of detergents.

      To whom correspondence may be addressed. Tel.: 517-355-9724 Fax: 517-353-9334 E-mail: [email protected]

      To whom correspondence may be addressed. Tel.: 517-355-0199 Fax: 517-353-9334 E-mail: [email protected]


      Modeling Protein–Protein and Protein–Nucleic Acid Interactions: Structure, Thermodynamics, and Kinetics

      Huan-Xiang Zhou , . Harianto Tjong , in Annual Reports in Computational Chemistry , 2008

      3.1 Electrostatic contribution

      It is well understood that hydrophobic interactions make favorable contributions to binding. However, the effects of electrostatic interactions are subtle. Neglecting conformational changes, the electrostatic contribution is given by

      where G el is the electrostatic free energy of each subunit (A or B) or the complex (AB), which can be calculated by solving the Poisson–Boltzmann (PB) equation. The subtlety of the electrostatic contribution can be appreciated by decomposing it into two components: the desolvation cost W desol and the solvent-screened interaction energy W int ( Figure 4.2 ). To obtain W desol , the electrostatic solvation energy of each subunit is calculated twice, first by itself and then in the presence of its partner, which has its partial charges zeroed out. The difference in the results between these two calculations gives the desolvation cost for that subunit, and adding the corresponding quantity for its partner gives W desol . The difference between W el and W desol comes from the interactions between the partial charges of the two subunits in the solvent environment.

      Figure 4.2 . Decomposition of the electrostatic contribution to binding affinity into desolvation cost and solvent-screened interaction. Interactions of protein charges with the solvent (represented by shadows around binding molecules) are indicated by outgoing arrows. Upon binding, the binding molecules are desolvated within their interface and charge–charge interactions, as indicated by a double-headed arrow, emerge.

      It is clear that W desol opposes binding. W int will favor binding when the charges on the two subunits have complementary charge distributions, which should be true in general. There W el consists of two opposite components. Whether electrostatic interactions make a net positive or net negative contribution to binding rests on the balance between the two components. In particular, the balance is very sensitive to how the boundary between the protein low dielectric and the solvent high dielectric is precisely specified. As shown on a large number of protein–protein and protein–RNA complexes [16–19] , when the dielectric boundary is chosen as the molecular surface (MS), as is often done in the literature, W desol outweighs W int , leading to net destabilization. However, when the dielectric boundary is switched to the van der Waals (vdW) surface, the situation is reversed and electrostatic stabilization is now predicted.

      How can one then decide on the choice of the dielectric boundary? One possibility is to benchmark PB calculations against explicit-solvent molecular dynamics (MD) simulations. Most of such efforts have been limited to small solute molecules [20–22] . However, it has been shown that the difference between MS and vdW results for electrostatic solvation energies depends on solute size [23] . Therefore parameterization on small solutes (either against explicit-solvent MD results or against experimental data) may not be reliable for calculating electrostatic contributions to protein–protein and protein–nucleic acid binding.

      One can benchmark PB calculations directly against experimental data on protein–protein and protein–nucleic acid binding affinities. Potentially one type of useful data is the dependence of binding affinities on salt concentration. The screening of electrostatic interactions by salts can be captured by the PB equation (it should be mentioned that salts can also specifically bind to proteins and nucleic acids such specific salt effects require special treatment). Unfortunately, it has been found that the screening effects predicted by MS and vdW calculations are essentially identical and thus cannot discriminate between the two choices of the dielectric boundary [16,18] . On the other hand, effects of mutations involving charged or polar residues have been found to have discriminating power, with experimental data favoring the vdW surface as the choice for the dielectric boundary [16–18] . Experimental data for mutational effects on binding affinity continue to accumulate in the literature [24,25] , providing opportunities for comprehensive benchmarking of PB calculation protocols.

      In the literature, the MS is still widely chosen as the dielectric boundary. The difference between this choice and the vdW surface is that, according to the latter protocol, the many crevices in the protein interior are treated as part of the solvent high dielectric. These crevices are not accessible to a spherical solvent used in defining the MS, and hence their being treated as part of the solvent dielectric is perceived as unrealistic or undesirable. However, this perception is open to question. Water molecules can access protein interiors, as demonstrated by many protein X-ray structures with water occupying interior positions, by the observation of positionally disordered water molecules in a hydrophobic cavity of interleukin 1b [26] ( Figure 4.3 ), and by molecular dynamics simulations [27] . In proteins like myoglobin and acetylcholinesterase (featuring a deeply buried active site connected to the exterior only through a narrow gorge), access by small molecules like water, made possible by the dynamics of the proteins, is essential for biological functions. We suggest that the vdW protocol provides a way to account for water access to protein interiors. Failure to account for this important property is perhaps the cause for overprediction of p K a shifts by the MS protocol (which is often “corrected” by increasing the protein dielectric constant to 20). In principle the vdW protocol can be mimicked by the MS protocol with appropriately reduced atomic radii. However, it has been found the precise amount of radius reduction varies from protein to protein and thus mimicking one protocol by the other appears to be a futile exercise [23] . We will come back to the debate between MS and vdW in Section 4.3 .

      Figure 4.3 . The presence of water molecules inside a hydrophobic cavity of interleukin 1b. The cavity is separated from the bulk solution according to the MS criterion but connected to the bulk solution according to the vdW criterion. When the three water molecules are moved from separate positions in the bulk solution to the configurations shown inside the cavity, the MS protocol predicts an increase of 0.9 kcal/mol in electrostatic free energy whereas the vdW protocol predicts a decrease of −2.2 kcal/mol.

      The generalized Born (GB) model has been developed as a fast substitute of the PB equation [28–31] . The GB model can be tailored to match PB results for electrostatic solvation energies obtained by either the MS or the vdW protocol. The errors of GB results in reproducing the PB counterparts are at least of the order of typical mutational effects on binding affinities. Therefore caution should be exercised when applying the GB model to calculate mutational effects.

      There is also progress in the opposite direction, i.e., toward more accurate modeling of electrostatic effects, by accounting for electronic polarization via quantum mechanical treatments [32,33] . Such treatments have not been used to directly predict the effects of mutations on the binding free energy, but it is already clear that electronic polarization can significantly influence electrostatic contributions to binding.

      Comparing PB or GB calculations against experimental data for mutational effects on binding affinity is premised on the assumption that the mutational effects are assumed to be dominated by electrostatic contributions. That is, possible contributions by hydrophobic interactions and by changes of conformational entropy are not taken into consideration.


      Videoni tomosha qiling: Получение кварцаSynthesis of silicon dioxide SiO2 (Iyul 2022).


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