Ma `lumot

2F: Termodinamika va XVFning oqsillar barqarorligi - biologiya


O'quv maqsadlari

  • Umumiy zaryad va o'ziga xos ion-ion juftlarini ajrating va ularning oqsil turg'unligidagi rolini umumlashtiring
  • N-metilatsetamid (NMA) tuzilishini chizib bering va nima uchun oqsil barqarorligida H bog'lanishining rolini o'rganishning foydali kichik molekula modeli ekanligini tushuntiring.
  • NMA ning vodorod bilan bog'langan dimerini suvdan qutbsiz muhitga o'tkazish uchun termodinamik tsiklni chizing. DG0 dan tsikldagi qadamlar va ushbu modelni oqsilgacha kengaytirish uchun, ko'milgan H bog'lanishining hosil bo'lishi oqsillarning katlanishiga olib kelishini taxmin qiling.
  • Past haroratli oqsil denatürasyonu, yuqori haroratli oqsil va DGo ning polar bo'lmagan yon zanjirlarni suvdan ko'proq polar bo'lmagan erituvchilarga o'tkazishi, oqsillar stabilitesidagi hidrofob ta'sirini qo'llab -quvvatlasa, tushuntiring.
  • Denaturantlar (karbamid, guanidin tuzlari) va stabilizatorlar (glitserin, ammoniy sulfat) ning oqsillarga ta'sirini tushuntirish uchun empirik Hofmeister seriyasi va reagentlarni oqsilning gidratlanish sohasiga majburiy bog'lanishi o'rtasidagi munosabatni umumlashtiring.
  • Suvda benzolning eruvchanligini oqsilni ochilishida hidrofob ta'sirining rolini o'rganish va oqsilning barqarorligini aniqlashda namuna sifatida, benzolni suvga o'tkazish uchun DG0, DH0, DS0 va DCp grafiklarini haroratning funktsiyasi sifatida izohlang.
  • yuqoridagi grafikdan tushuntiring, agar benzolning suvga o'tishining termodinamik parametrlaridagi tendentsiyalar oqsillarning kuzatiladigan oqsillarning haroratga bog'liqligini/barqarorligini bashorat qiladimi?
  • Polar bo'lmagan molekulalarning suvga o'tishi uchun kuzatilgan DCp ning molekulyar talqinini bering.
  • Zanjirli konformatsion entropiyani ta'riflang, uni bitta va ikkita zanjirli amfifillardagi asil yon zanjirlaridagi harorat o'zgarishi bilan bog'lang va oqsil barqarorligida uning rolini tasvirlab bering.
  • Asn -Ala mutatsiyasining oqsil tarkibidagi kuzatiladigan beqarorlashtiruvchi ta'siri uchun berilgan bir nechta tushuntirishlar, bu kuzatuvlar uchun.
  • oqsil barqarorligiga asosiy hissa qo'shuvchilarning (molekulalararo va kuchlararo ta'sirlar) kattaligi va yo'nalishini grafik jihatdan umumlashtiring.

Ma'lumki, oqsillar barqaror emas va har xil kattalikdagi hissa qo'shishi kerak, bu esa oqsillarga fiziologik sharoitda chegaraviy barqarorlikni beradi. Yangi ma'noda aniqlangan gidrofobik o'zaro ta'sir barqarorlikda katta rol o'ynashi kerak. Bundan tashqari, oqsillar yo'qolgan denatura holatiga nisbatan juda ko'p to'planganligi sababli, London kuchlari ham muhim rol o'ynashi kerak. (Yodingizda bo'lsin, dispersiya kuchlari qisqa masofali va eng yaqin qadoqlash sharoitida eng ahamiyatli bo'ladi.) Qarama -qarshi katlama - bu yuqorida tavsiflangan konformatsion zanjir entropiyasi. Proteinlar shunchalik barqaror bo'lgani uchun, hatto bitta bog'lanmagan ko'milgan ionli yon zanjir yoki oqsil tarkibidagi 1-2 ta bog'lanmagan H bog'lanish donorlari va akseptorlari ham mahalliy tuzilmani "ochish" uchun etarli bo'lishi mumkin, bu esa denatura holatiga olib keladi.

Eskiz: Odam gemoglobinining tuzilishi. Oqsillar a va b kichik bo'linmalari qizil va ko'k rangda, tarkibida temir bo'lgan gem guruhlari yashil rangda. PDB dan: 1GZX. (GNU; Proteopediya gemoglobin).


2 -BOB - PROTEIN TUZILISHI

2F bo'limi uchun o'quv maqsadlari/vazifalari: Darsdan va o'qishdan so'ng talabalar buni qila oladilar.

  • Umumiy zaryad va o'ziga xos ion-ion juftlarini ajrating va ularning oqsil turg'unligidagi rolini umumlashtiring
  • N-metilatsetamid (NMA) tuzilishini chizing va oqsil barqarorligida H bog'lanishining rolini o'rganish nega foydali kichik molekula modeli ekanligini tushuntiring.
  • NMA ning vodorod bilan bog'langan dimerini suvdan qutbsiz muhitga o'tkazish uchun termodinamik tsiklni chizing. DG0 dan tsikldagi qadamlar va ushbu modelni oqsilgacha kengaytirish uchun, ko'milgan H bog'lanishining hosil bo'lishi oqsillarning katlanishiga olib kelishini taxmin qiling.
  • Past haroratli oqsil denatürasyonu, yuqori haroratli oqsil va DGo ning polar bo'lmagan yon zanjirlarni suvdan ko'proq polar bo'lmagan erituvchilarga o'tkazishi, oqsillar stabilitesidagi hidrofob ta'sirini qo'llab -quvvatlasa, tushuntiring.
  • Denaturantlar (karbamid, guanidin tuzlari) va stabilizatorlarning (glitserin, ammoniy sulfat) oqsillarga ta'sirini tushuntirish uchun empirik Hofmeister seriyasi va reagentlarni oqsilning gidratlanish sohasiga majburiy bog'lanishi o'rtasidagi munosabatni umumlashtiring.

Suvda benzolning eruvchanligini oqsilni ochilishida hidrofob ta'sirining rolini o'rganish uchun va oqsilning turg'unligidan xulosa qilib, benzolni suvga o'tkazish uchun DG0, DH0, DS0 va DCp grafiklarini haroratning funktsiyasi sifatida izohlang.

yuqoridagi grafikdan tushuntiring, agar benzolning suvga o'tishining termodinamik parametrlaridagi tendentsiyalar oqsillarning kuzatiladigan oqsillarning haroratga bog'liqligini/barqarorligini bashorat qiladimi?

Polar bo'lmagan molekulalarning suvga o'tishi uchun kuzatilgan DCp ning molekulyar talqinini bering.

Zanjirli konformatsion entropiyani ta'riflang, uni bitta va ikkita zanjirli amfifillardagi asil yon zanjirlaridagi harorat o'zgarishi bilan bog'lang va oqsil barqarorligida uning rolini tasvirlab bering.

Asn -Ala mutatsiyasining oqsil tarkibidagi kuzatiladigan beqarorlashtiruvchi ta'siri uchun berilgan bir nechta tushuntirishlar, bu kuzatuvlar uchun.

oqsil barqarorligiga asosiy hissa qo'shuvchilarning (molekulalararo va kuchlararo ta'sirlar) kattaligi va yo'nalishini grafik jihatdan umumlashtiring.


F1. Proteinlar barqarorligiga kirish

Bu material oson emas va, ehtimol, butun kitobning eng intellektual qiyinligi. Ushbu sharhning aksariyati Ken Dill, Biokimyo, 29, 7133-7155 (1990) maqolasidan olingan. Protein, Biokimyo tomonidan yozilgan, H bog'lanishlarining oqsillarni yig'ish va turg'unlikdagi roli borasida ancha boshqacha xulosaga kelgan yangi tahlil. 40, pg 310 (2001), oxirida muhokama qilinadi.

Xulosa qilib aytadigan bo'lsak, endi hidrofob ta'sir ham, H aloqalari ham oqsillarning burilishini qo'zg'atadi va oqsilning barqarorligiga yordam beradi. H ulanishining kichik modelli donorlari/aktseptorlari va hidrofob molekulalarini suvdan qutbsiz erituvchilarga o'tkazilishi bo'yicha olib borilgan tadqiqotlar natijalariga ko'ra, H bog'lanishining o'zaro ta'siri (shuningdek, ionli -ionli o'zaro ta'sirlar) ham oqsillarning buklanishiga olib kelmaydi. . Aksincha, mahalliy holatni barqarorlashtirishga eng katta hissa qo'shganlar oqsilning zich o'ralgan atomlari orasidagi hidrofob ta'sir va van der Valsning o'zaro ta'siri. Biroq, mutant oqsillarning saytga xos mutagenez orqali o'tkazilgan so'nggi tadqiqotlari (Pace) dan ma'lum bo'lishicha, H bog'lanishlari oqsillarning qatlanishi va turg'unligiga sezilarli hissa qo'shadi va mahalliy holatning barqarorligiga hidrofob ta'siridan ko'ra ko'proq hissa qo'shishi mumkin. Katlanishga qarshi bo'lgan asosiy omil - bu zanjirli konformatsion entropiya. Bu ijobiy va manfiy omillar oqsillarning katlanishini ta'minlaydigan kichik salbiy DG ni tashkil qiladi, bu normal haroratda mahalliy oqsilning chegaraviy barqarorligini bildiradi.

Qanday turdagi molekulalararo kuchlar oqsil ichida va oqsillar va hal qiluvchi molekulalari o'rtasida harakat qilishi mumkin, bu oqsilning o'z -o'zidan uch o'lchovli 3D tuzilishga aylanishiga olib keladi? Bu kuchlar uzoq masofali (ion-ion, ion dipol yoki dipol-dipol) yoki qisqa diapazonli (van der Vals itaruvchi va jozibali kuchlar) bo'lishi mumkin. O'zaro ta'sirlar mahalliy bo'lishi mumkin (chiziqli ketma -ketlikdagi qo'shni aminokislotalar o'rtasida) yoki lokal bo'lmagan (chiziqli ketma -ketlikda ajratilgan, lekin 3D fazoda bir -biriga yaqin bo'lgan ketma -ketliklar o'rtasida). Mahalliy oqsilning uchinchi darajali tuzilishini nima barqarorlashtirishi haqida, oqsillarni oqsilni ochuvchi yoki denatura qiluvchi agentlarga yuborish orqali olish mumkin. Bunday agentlarga haddan tashqari pH, ba'zi tuz eritmalari yoki organik erituvchilarning yuqori konsentratsiyasi va haroratning haddan tashqari o'zgarishi kiradi. Bunday tajribalar shuni ko'rsatadiki, mahalliy oqsillar faqat bir oz barqaror (taxminan 0,4 kJ/mol aminokislota - yoki 10 000 kkal/mol molekulyar og'irlikdagi protein uchun - taxminan 100 aminokislotalar). Biz molekulalararo kuchlarning har xil turlarini (ion-ion, H-bog'lanishlar, van-der-Vals va hidrofob ta'sir) alohida ko'rib chiqamiz va ularning har biri oqsillarni yig'ish uchun muhim harakatlantiruvchi kuch ekanligini so'raymiz.

Rasm: oqsillarni yig'ish uchun DG ga qo'shilgan hissalarni ko'rsatadigan diagramma.

Ushbu bobning ko'p qismi H bog'lanish va hidrofob ta'siriga bag'ishlanadi. Har qanday biokimyo kursining mavzusi shundan iboratki, agar siz kichik molekulalarning o'zaro ta'sirini tushunsangiz, bu ma'lumotni oqsillar kabi katta molekulalarni tushunishda qo'llashingiz mumkin. Oqsillar ichidagi H bog'lanishlari, ko'pincha oqsilning ko'proq hidrofob ichki qismiga ko'milganligi, oqsillarning burilishini qo'zg'atadi, biz avval N-metilatsetamidning suvda va qutbsiz erituvchida H bog'lanish hosil bo'lishining termodinamikasini o'rganamiz. Oqsilning polar bo'lmagan markaziga polar bo'lmagan yon zanjirlarni ko'mish orqali gidrofob ta'sirining oqsillarning burilishini qo'zg'atishini tushunish uchun biz suvda benzolning eruvchanligi termodinamikasini o'rganamiz. Oxirgi tadqiqotlarda aminokislotalar holatida H mutanosibligi va hidrofob ta'sirining buklanish va oqsillar turg'unligiga ta'sirini aniqlaydigan mutantlar yaratilishi nazarda tutilgan.

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Xulosa

Gp41 konvertli oqsil virusli va hujayrali membranalarning birlashishini rag'batlantirish orqali primat immunitet tanqisligi viruslarining maqsadli hujayralarga kirishiga vositachilik qiladi. Gp41 ektodomain yadrosining tuzilishi bir xil spiral soch qisqichlarining trimerini ifodalaydi, bunda uchta amino-terminalli spiraldan tashkil topgan markaziy trimerik spiral uchta antiparallel karboksil-terminalli spiralning tashqi qavatiga o'ralgan. Bu termoyadroviy faol gp41 konformatsiyasining tetiklantiruvchi shakllanishi membrananing yaqin joylashishiga olib keladi va shu tariqa lipidli ikki qatlamli sintez uchun faollashuvchi energiya to'sig'ini yengadi. Biz rekombinant trimerik N34 (L6) C28 modeli yordamida simian immunitet tanqisligi virusi (SIV) gp41 yadrosining katlanadigan termodinamikasining batafsil tavsifini taqdim etamiz. Diferensial skanerlash kalorimetriyasi va denaturant ta'sirida va termal ochilish bo'yicha o'tkaziladigan spektroskopik tajribalar shuni ko'rsatadiki, uchta 68 ta qoldiq N34 (L6) C28 peptidlarining soch trimerlariga birikish erkin energiyasi pH 7.0 va 25 ° C da 76 kJ mol -1 ga teng. fiziologik tamponda. Bilan bog'liq entalpiya o'zgarishi, gHyaroqsiz, 177 kJ mol -1, ochilish entropiyasi esa ΔSyaroqsiz, 0,32 kJ K -1 mol -1. Maksimal barqarorlik harorati 20 ° C ga yaqin. Issiqlik sig'imining oshishi ~ 9 kJ K -1 mol -1 (p45 J K -1 mol qoldig'i -1), bu trimerning kengaytirilgan va to'liq gidratlangan polipeptid zanjiriga ochilishi uchun kutilganidan past. N-terminalli trimerik o'ralgan-sarg'ish interfeysga ko'milgan Thr582 yoki Thr586 qutbli qoldiqlarini izolösin bilan almashtirish 30-35 kJ mol-1 trimerining mustahkamlanishiga olib keladi. O'rta bobinli bitta nuqtali mutatsiyalar gp41 yadro tuzilishini kuchli tarzda barqarorlashtiradi. Bu termodinamik xarakteristikalar gp41 konvertidagi oqsilni membranali termoyadroviy jarayonida uning termoyadroviy faol konformatsiyasiga qaytarilishi uchun muhim bo'lishi mumkin.


F9. Protein barqarorligi va molekulyar orbitallar

Protein tuzilishi va barqarorligining asosini tashkil etuvchi energetikaning to'liq tavsifi kvant mexanik ta'sirini o'z ichiga oladi. Kvant mexanik hisob -kitoblarni katta molekulalarda bajarish qiyin bo'lgani uchun, ular batafsil o'rganilmagan. Oxirgi ishlar oqsillarni elektron delokalizatsiyasi orqali stabillashtiradigan proksimal molekulyar orbitallarning bir -biriga o'xshashligiga asoslangan oqsillar barqarorligining yangi manbasini tushunishga olib keldi. Barqarorlikni oshiradigan elektron delokalizatsiyasining ikkita keng tarqalgan misoli - rezonans va giperkonjugatsiya. Rezonans, past energiyali molekulyar bog'lanish orbitallarini hosil qilish uchun qo'shni atomli p orbitallarning bir -birining ustiga chiqishi orqali elektronlarning delokalizatsiyasini o'z ichiga oladi. Uchinchi darajali karbokatlarning ikkilamchi yoki birlamchi bilan solishtirganda barqarorligini tushuntirish uchun giperkonjugatsiya ishlatilganini eslaysiz. Oddiy tushuntirishga elektron donorlik va qo'shni metil guruhlaridan itarish kiradi, bu esa karbokatsiya zaryadini samarali kamaytiradi. Aniqroq tushuntirish, molekulyar orbitallarning, xususan, metil guruhining C-H rishtalaridan birining C-H s orbitalining bir-biri bilan to'ldirilishini o'z ichiga oladi.

Orbital qoplama vodorod aloqalarining o'zaro ta'sirini hisobga olishi ajablanarli emas (vodorod aloqasi donorlari va akseptorlarining qisman zaryadlari o'rtasidagi tortishuvlarning soddalashtirilgan tushuntirishidan farqli o'laroq). Bartlett va boshqalar (2010) yaqinda oqsil tuzilmalarini barqarorlashtiradigan elektron delokalizatsiyasini o'z ichiga olgan ikki turdagi orbital bir -biriga o'xshashliklarni tasvirlab berishdi. Molekulyar orbital ta'rifi shuni ko'rsatadiki, amid bog'laridagi kislorod (vodorod aloqasi donori) bo'yicha ikkita yolg'iz juftlik farq qiladi. Bir juftlik, ns (pastdagi a -rasm) 60% s belgi bilan bog'lanmagan orbitalda, ikkinchisi (pastdagi b -rasm) taxminan 100% p belgidan iborat. Quyidagi c -rasmda ko'rsatilishicha, aminokislotaning karbonil kislorodi va aminokislotaning H+amid kislotasi o'rtasidagi vodorod aloqasi kvant mexanik jihatdan ns ning kislorod bilan bog'lanmagan orbitalining bir -biriga yopishishi (ya'ni delokalizatsiyasi) sifatida tasvirlanishi mumkin. NH orbitaliga qarshi s * antibonding. Karbonil O np elektronlari haqida nima deyish mumkin? Ular ikkita aminokislotalar ikkilamchi tuzilmalarning bir qismi sifatida proksimal bo'lganida, np orbitalining ith+1 karbonil guruhidagi antioksidlovchi p * orbital bilan bir -biriga to'g'ri kelishi bilan oqsillarga yangi turdagi barqarorlashtiruvchi ta'sir ko'rsatadigan dalillarni topdilar. alfa va 310 spiral, shuningdek b o'ralgan b varaqlarda.

(a) amid kislorodga boy bo'lgan yagona juftlik. (b) amid kislorodning p-ga boy yakka juftligi. (c) ns to σ*: a -spiraldagi vodorod aloqasi. (d) np to p *: n to p * a -spiraldagi o'zaro ta'sir. Macmillan Publishers Ltd: Tabiiy kimyoviy biologiya ruxsati bilan qayta nashr etilgan. Bartlett, G. va boshqalar. n to pi* proteindagi o'zaro ta'sirlar. 6, 615 -bet (2010)

Qaysi phi-psi burchaklari etarlicha yaqinlashishga imkon berishini tekshirish uchun np ga p * shovqinlari, tergovchilar AcAla4NHMe ning mumkin bo'lgan muvofiqligi bo'yicha kvant mexanik hisob -kitoblarni amalga oshirishdi. np ga p * mumkin edi (quyida A rasmiga qarang). Bu hisob -kitoblar shuni ko'rsatdiki, bu o'zaro ta'sir d ≤ 3.2 va 99 ≤ ≤ ≤ 119 qiymatlari uchun mumkin edi (a -rasm). 3,2 angstromdan kam masofalar, van der Valsning C va O sirtlari qo'shni karbonillarda bir -biriga to'g'ri keladi, bu o'zaro ta'sir kvant mexanik va klassik vositachilik emasligini ko'rsatadi. Keyin ular ma'lum oqsillarning yuqori aniqlikdagi rentgen tuzilmalari uchun d va q qiymatlarini hisobladilar. Ramachandran uchastkasidagi qizil soyalar, kerakli geometriyaga mos keladigan haqiqiy oqsillarda phi/psi mintaqalari ostidagi s rasmda. np ga p * bir -biriga mos keladi. Demak, bu geometriya oqsillarda, ayniqsa yuqorida tasvirlangan ikkilamchi tuzilmalarda ko'p.

(a) dihedral burchaklarning ta'riflari φ (C'i 1 Ni Ca i Ci) va ψ (NiCai CiiNi+1), masofa (d) va tekislik burchagi θ. Kristallografik tahlillarda n dan p * gacha bo'lgan o'zaro ta'sir mezonlari: d ≤ 3.2 <99 le le 119 . (b) energiyani ko'rsatadigan hisoblash ma'lumotlari. Macmillan Publishers Ltd: Tabiiy kimyoviy biologiya ruxsati bilan qayta nashr etilgan. Bartlett, G. va boshqalar. n to pi* proteindagi o'zaro ta'sirlar. 6, 615 -bet (2010)


4.6: Oqsillar barqarorligida termodinamika va XVF

  • Genri Yakubovski tomonidan qo'shilgan
  • Sent -Benedikt kolleji professori (kimyo). Jon universiteti
  • Umumiy zaryad va o'ziga xos ion-ion juftlarini ajrating va ularning oqsil turg'unligidagi rolini umumlashtiring
  • N-metilatsetamid (NMA) tuzilishini chizing va oqsil barqarorligida H bog'lanishining rolini o'rganish nega foydali kichik molekula modeli ekanligini tushuntiring.
  • NMA ning vodorod bilan bog'langan dimerini suvdan qutbsiz muhitga o'tkazish uchun termodinamik tsiklni chizing. DG0 dan tsikldagi qadamlar va ushbu modelni oqsilgacha kengaytirish uchun, ko'milgan H bog'lanishining hosil bo'lishi oqsillarning katlanishiga olib kelishini taxmin qiling.
  • Past haroratli oqsil denatürasyonu, yuqori haroratli oqsil va DGo ning polar bo'lmagan yon zanjirlarni suvdan ko'proq polar bo'lmagan erituvchilarga o'tkazishi, oqsillar stabilitesidagi hidrofob ta'sirini qo'llab -quvvatlasa, tushuntiring.
  • Denaturantlar (karbamid, guanidin tuzlari) va stabilizatorlar (glitserin, ammoniy sulfat) ning oqsillarga ta'sirini tushuntirish uchun empirik Hofmeister seriyasi va reagentlarni oqsilning gidratlanish sohasiga majburiy bog'lanishi o'rtasidagi munosabatni umumlashtiring.
  • Suvda benzolning eruvchanligini oqsilni ochilishida hidrofob ta'sirining rolini o'rganish va oqsilning barqarorligini aniqlashda namuna sifatida, benzolni suvga o'tkazish uchun DG0, DH0, DS0 va DCp grafiklarini haroratning funktsiyasi sifatida izohlang.
  • yuqoridagi grafikdan tushuntiring, agar benzolning suvga o'tishining termodinamik parametrlaridagi tendentsiyalar oqsillarning kuzatiladigan oqsillarning haroratga bog'liqligini/barqarorligini bashorat qiladimi?
  • Polar bo'lmagan molekulalarning suvga o'tishi uchun kuzatilgan DCp ning molekulyar talqinini bering.
  • Zanjirli konformatsion entropiyani ta'riflang, uni bitta va ikkita zanjirli amfifillardagi asil yon zanjirlaridagi harorat o'zgarishi bilan bog'lang va oqsil barqarorligida uning rolini tasvirlab bering.
  • Asn -Ala mutatsiyasining oqsil tarkibidagi kuzatiladigan beqarorlashtiruvchi ta'siri uchun berilgan bir nechta tushuntirishlar, bu kuzatuvlar uchun.
  • oqsil barqarorligiga asosiy hissa qo'shuvchilarning (molekulalararo va kuchlararo ta'sirlar) kattaligi va yo'nalishini grafik jihatdan umumlashtiring.

Ma'lumki, oqsillar barqaror emas va har xil kattalikdagi ko'p hissa qo'shilishi kerak, bu esa oqsillarga fiziologik sharoitda chegaraviy barqarorlikni beradi. Yangi ma'noda aniqlangan gidrofobik o'zaro ta'sir barqarorlikda katta rol o'ynashi kerak. Bundan tashqari, oqsillar yo'qolgan denatura holatiga nisbatan juda ko'p to'planganligi sababli, London kuchlari ham muhim rol o'ynashi kerak. (Yodingizda bo'lsin, dispersiya kuchlari qisqa masofali va eng yaqin qadoqlash sharoitida eng ahamiyatli bo'ladi.) Qarama -qarshi katlama - bu yuqorida tavsiflangan konformatsion zanjir entropiyasi. Proteinlar bir xil darajada barqaror bo'lgani uchun, hatto bitta bog'lanmagan ko'milgan ionli yon zanjir, yoki 1-2 ta bog'lanmagan ko'milgan H bog'lanishli donorlar va oqsildagi akseptorlar ham mahalliy tuzilmani "denaturatsiyalangan" holatga olib kelish uchun etarli bo'lishi mumkin.

Eskiz: Odam gemoglobinining tuzilishi. Proteinlar va alfa va beta-bo'linmalar qizil va ko'k rangda, tarkibida temir bo'lgan gem guruhlari yashil rangda. PDB dan: 1GZX ​. (GNU Proteopediyasi Gemoglobin).


F9. Protein barqarorligi va molekulyar orbitallar

  • Genri Yakubovski tomonidan qo'shilgan
  • Sent -Benedikt kolleji professori (kimyo). Jon universiteti

Protein tuzilishi va barqarorligining asosini tashkil etuvchi energetikaning to'liq tavsifi kvant mexanik ta'sirini o'z ichiga oladi. Kvant mexanik hisob -kitoblarni katta molekulalarda bajarish qiyin bo'lgani uchun ular batafsil o'rganilmagan. Oxirgi ishlar oqsillarni elektron delokalizatsiyasi orqali stabillashtiradigan proksimal molekulyar orbitallarning bir -biriga o'xshashligiga asoslangan oqsillar barqarorligining yangi manbasini tushunishga olib keldi. Barqarorlikni oshiradigan elektron delokalizatsiyasining ikkita keng tarqalgan misoli - rezonans va giperkonjugatsiya. Rezonans, past energiyali molekulyar bog'lanish orbitallarini hosil qilish uchun qo'shni atomli p orbitallarning bir -birining ustiga chiqishi orqali elektronlarning delokalizatsiyasini o'z ichiga oladi. Uchinchi darajali karbokatlarning ikkilamchi yoki birlamchi bilan solishtirganda barqarorligini tushuntirish uchun giperkonjugatsiya ishlatilganini eslaysiz. Oddiy tushuntirishga elektron donorlik va qo'shni metil guruhlaridan itarish kiradi, bu esa karbokatsiya zaryadini samarali kamaytiradi. Aniqroq tushuntirish, molekulyar orbitallarning, xususan, metil guruhining C-H rishtalaridan birining C-H s orbitalining bir-biri bilan to'ldirilishini o'z ichiga oladi.

Rasm: Giperkonjugatsiya orqali karbokatsiyalarni barqarorlashtirish

Orbital qoplama vodorod aloqalarining o'zaro ta'sirini hisobga olishi ajablanarli emas (vodorod aloqasi donorlari va akseptorlarining qisman zaryadlari o'rtasidagi tortishuvlarning soddalashtirilgan tushuntirishidan farqli o'laroq). Bartlett va boshqalar (2010) yaqinda oqsil tuzilmalarini barqarorlashtiradigan elektron delokalizatsiyasini o'z ichiga olgan ikki turdagi orbital qoplamalarning ta'rifini berishdi. Molekulyar orbital ta'rifi shuni ko'rsatadiki, amid bog'laridagi kislorod (vodorod aloqasi donori) bo'yicha ikkita yolg'iz juftlik farq qiladi. Bir juftlik, ns (pastdagi a -rasm) 60% s belgi bilan bog'lanmagan orbitalda, ikkinchisi (pastdagi b -rasm) taxminan 100% p belgidan iborat. Quyidagi c -rasmda ko'rsatilishicha, aminokislotaning karbonil kislorodi va aminokislotaning H+amid kislotasi o'rtasidagi vodorod aloqasi kvant mexanik ma'noda ns ning kislorod bilan bog'lanmagan orbitalining bir -biriga yopishishi (ya'ni delokalizatsiyasi) sifatida tasvirlanishi mumkin. NH orbitaliga qarshi s * antibonding. Karbonil O np elektronlari haqida nima deyish mumkin? Ular ikkita aminokislotalar ikkilamchi tuzilmalarning bir qismi sifatida proksimal bo'lganida, np orbitalining ith+1 karbonil guruhidagi antioksidlovchi p * orbital bilan bir -biriga to'g'ri kelishi bilan oqsillarga yangi turdagi barqarorlashtiruvchi ta'sir ko'rsatadigan dalillarni topdilar. alfa va 310 spiral, shuningdek b o'ralgan b varaqlarda.

Shakl: H-bog'lanishlarda orbital ishtiroki va alfa spirallaridagi n to pi* o'zaro ta'siri

(a) amid kislorodga boy bo'lgan yagona juftlik. (b) amid kislorodning p-boy yakka juftligi. (c) ns to & sigma*: a -spiraldagi vodorod aloqasi. (d) np to p *: n to p * a -spiraldagi o'zaro ta'sir. Macmillan Publishers Ltd: Tabiiy kimyoviy biologiya ruxsati bilan qayta nashr etilgan. Bartlett, G. va boshqalar. n to pi* proteindagi o'zaro ta'sirlar. 6, 615 -bet (2010)

Qaysi phi-psi burchaklari etarlicha yaqinlashishga imkon berishini tekshirish uchun np ga p * shovqinlari, tergovchilar AcAla4NHMe ning mumkin bo'lgan muvofiqligi bo'yicha kvant mexanik hisob -kitoblarni amalga oshirishdi. np ga p * mumkin edi (quyida A rasmiga qarang). Hisob -kitoblar shuni ko'rsatdiki, bu o'zaro ta'sir d & le 3.2 � va 99 � & le & theta & le 119 � qiymatlari uchun mumkin edi (pastdagi rasm). 3,2 angstromdan kam masofalar, van der Valsning C va O sirtlari qo'shni karbonillarda bir -biriga to'g'ri keladi, bu o'zaro ta'sir kvant mexanik va klassik vositachilik emasligini ko'rsatadi. Keyin ular ma'lum oqsillarning yuqori aniqlikdagi rentgen tuzilmalari uchun d va q qiymatlarini hisobladilar. Ramachandran uchastkasidagi qizil soyalar, kerakli geometriyaga mos keladigan haqiqiy oqsillarda phi/psi mintaqalari ostidagi rasmda. np ga p * bir -biriga mos keladi. Demak, bu geometriya oqsillarda, ayniqsa yuqorida tasvirlangan ikkilamchi tuzilmalarda ko'p.

Rasm: Ramachandran n -pi* shovqinlarining uchastkalari. (a) dihedral burchaklar va phi ta'riflari (C va bosh 򟿣 �Ni �C & alfa i �C va bosh) va psi (Ni �C & alfa �C va bosh �Ni+1), masofa (d) va tekislik. Kristallografik tahlillarda n -p * o'zaro ta'sirining mezonlari: d & le 3.2 � 99 � & le & theta & le 119 �. (b) energiyani ko'rsatadigan hisoblash ma'lumotlari. Macmillan Publishers Ltd: Tabiiy kimyoviy biologiya ruxsati bilan qayta nashr etilgan. Bartlett, G. va boshqalar. n to pi* proteindagi o'zaro ta'sirlar. 6, 615 -bet (2010)


Barcha hujayra ichidagi jarayonlarda oqsil tuzilishi va dinamikasi makromolekulyar to'planish (MC) ta'siriga uchraydi. Bu erda har xil turdagi va o'lchamdagi MC agentlarining model oqsiliga ta'siri Bacillus subtilis Sovuq zarba oqsili va#x02005B (BsCspB) termal va kimyoviy denaturatsiya paytida har tomonlama o'rganilgan. Biz doimiy ravishda aniq barqarorlikni aniqlaymiz BsCspB MC kontsentratsiyasiga bog'liq, lekin ishlatilgan MC agentining yopishqoqligi, qutbliligi yoki hajmiga bog'liq emas. Bu umumiy stabilizatsiya NMR spektroskopiyasi yordamida, kimyoviy siljish (CS) buzilishlari va molekula ichidagi vodorod va bog'lanish tarmoqlarini kuzatish, shuningdek amid protonlarini hal qiluvchi protonlar bilan almashishdan himoyalash orqali dekodlangan. MC mavjud bo'lganda CS va vodorod va#x02010 ulanish tarmoqlariga tizimli ta'sir ko'rsatilmagan bo'lsa, biz pastadirli hududlarda almashinuvning sezilarli kamayishini aniqladik. BsCspB. Biz shunday xulosaga keldikki, erituvchi protonlarga kirishning pasayishi MC ostida kuzatilgan oqsil barqarorligining oshishi uchun asosiy parametrdir.

Hamma tirik organizmlarda oqsillarning yig'ilishi va funktsiyasi 400 g gacha bo'lgan makromolekulalarni o'z ichiga olgan, odamlar gavjum bo'lgan muhitda sodir bo'ladi. faqat tamponlash agenti va bir nechta ligandlar yoki reaktivlardan iborat bo'lib, natijada kuchli muhit suyultiriladi. Tabiiyki, bu shartlar hujayra ichidagi muhitni aks ettirmaydi, chunki oqsillarning strukturaviy, dinamik va funktsional xususiyatlari konformatsiya va barqarorlikning bir daqiqalik o'zgarishlariga bog'liq. Bundan tashqari, o'zaro ta'sirning ma'lum joylariga cheklangan kirishni kimyoviy muhitning ozgina o'zgarishi osongina buzishi mumkin. Binobarin, in vivo jonli sharoitda uchraydigan biologik mexanizmlar va konformatsion ansambllar in vitro stsenariylardan farq qiladi.2 Vivo jonli ravishda sodir bo'layotgan barcha jarayonlarni har tomonlama tushunish nafaqat biokimyoviy nuqtai nazardan, balki tibbiy nuqtai nazardan ham muhimroqdir. , masalan, amyotrofik lateral skleroz (ALS), Altsgeymer va Parkinson kasalligi yoki qandli diabet kabi kasalliklarni keltirib chiqaradigan oqsil agregatlari va amiloid tuzilmalar shakllanishini hisobga olgan holda 𠁒.

Proteostazni chuqur tushunishning kaliti - bu, birinchi navbatda, odamlar gavjum bo'lgan uyali ichki makonni dekodlash. MC sohasidagi so'nggi o'zgarishlar, faqat entropik ajratilgan hajm (EV) effekti nuqtai nazaridan, EV effektiga hissa qo'shadigan yoki kamaytiradigan entalpik “soft ” yoki ȁximiyaviy o'zaro ta'sirlarning murakkab o'zaro ta'siriga o'tdi. 3 Bundan tashqari, zaryad va#x02013 zaryadli o'zaro ta'sirlar4 va oqsil yuzalarining roli, ayniqsa, “in �ll ” tajribalari5 va simulyatsion tadqiqotlar6 nuqtai nazaridan muhokama qilindi, ular hujayralarga nuqtai nazarni oqsillarga to'la sumkalar va hujayralarga aylantirdi. x0201cprotein tomchilari ”.7 Biroq, 1981 yildan buyon makromolekulyar to'planish (MC) sohasidagi yutuqlarga qaramay, oqsil tuzilishiga tegishli in vitro ma'lumotlarini to'g'ridan -to'g'ri tarjima qilishga imkon beradigan hech qanday tub tushuncha yo'q. dinamikasi va funktsiyasi in vivo jonli stsenariylarda, 30 yil oldin “macromoecular crowding ” atamasini kiritgan Minton yaqinda ta'kidlaganidek.

Protein ansambllarini har tomonlama o'rganish uchun zarur bo'lgan aniqlikdagi in vivo tajribalar kamdan -kam uchraydi va molekulyar ta'sirlarning individual hissasini ajratib bo'lmaydi, shuning uchun biz bu erda olomon hujayra ichidagi muhitni taqlid qiladigan ortogonal tajribalarga asoslangan tizimli pastki yondashuvni taqdim etamiz. Biz MC ning kattaligiga ham, qutblanishiga ham ta'sir qilish uchun har xil o'lchamdagi polimerik to'kish vositalarini dekstran, shuningdek poli (etilen glikol) (PEG) dan foydalanishga qaror qildik. PEGning inert to'plash vositasi sifatida hidrofobik yon ta'siriga kelsak, bizning modelimiz oqsillari, masalan, sitoxrom va#x02005v 10a va#x02014, PEG va hidrofob sirt yamoqlari orasidagi o'ziga xos ta'sirga moyil emas (pastga qarang). Xuddi shunday, PEG RNase 𠁚 turg'unligini oshirishga qodir, lekin bu oqsil bilan to'g'ridan -to'g'ri ta'sir o'tkazmaydi. 10b Sintetik polimerlar yordamida ham sterik, ham elektrostatik ta'sirlarni kuzatish mumkin, shu bilan birga oqsil yig'uvchilarni ishlatganda yuzaga keladigan o'ziga xos o'zaro ta'sirlar bundan mustasno. va eksperimental natijalarni bir xil ma'noda bizning tadqiqotimizning markazini tashkil etuvchi MC xususiyatlariga tayinlash mumkin. Bu tizimli eksperimental yondashuvning ahamiyati katta, chunki MC sohasidagi ko'plab hissalar, birinchi navbatda, faqat bitta turdagi to'plash vositasidan foydalanish, ikkinchidan, bitta eksperimental texnika yordamida. muqobil usullar natijalari bilan solishtirishga ruxsat bermaydi va uchinchidan, juda murakkab tizimlarga e'tibor qaratadi.2e, 11

Bizning tadqiqotimiz tizimli yondashuvni ta'minlaydi Bacillus subtilis Sovuq zarba oqsili va#x02005B (BsCspB), 12a, 12b, bu katlama nuqtai nazaridan ochiladigan o'tish davriga qadar ikkita ‐ davlat tizimining yorqin namunasidir. konsentrasiyalarning keng diapazonida mavjud bo'lib, turli xil texnikalar yordamida kuzatiladi. Bu bizga konvergent tarzda MC effektining termodinamik kelib chiqishini tushunishimizga imkon berdi.

Karbamid borligi bilan kimyoviy denatürasyondan boshlab, biz mahalliy davlatning kuchli barqarorligini topdik BsCspB, floresan spektroskopiya yordamida kuzatiladigan PEG8 qo'shilganda (Qo'llab -quvvatlovchi ma'lumotdagi   1 va#x02009A -rasm, jadval va#x02005S1 rasm). 20 va#x02009% da (w/v) Eritmada PEG8, stabilizatsiyasi BsCspB shu qadar ajoyibki, karbamid va PEG8 ning eruvchanligi oralig'ida to'liq denaturatsiyalangan holatga etib bo'lmaydi ( S1 -rasm). Qizig'i shundaki, PEG1 qo'shilishi barqarorlashdi BsCspB, ishlatilgan model oqsiliga o'xshash molekulyar og'irlikdagi MC agenti bo'lgan PEG8 bilan bir xil darajada (Shakl   1 𠂛, Jadval va#x02005S1). Barqarorlikning bu o'sishi PEG8 ta'siriga teng bo'ladi, bu esa barqarorlikni ko'rsatadi BsCspB berilgan yopishqoqlikdan mustaqildir, bu PEG1 ni PEG8 bilan solishtirganda besh barobar farq qiladi va 20% (w/v) (Jadval va#x02005S2).

Kimyoviy ochilish BsA), B), E)   karbamid yoki C), D), F)  GdmCl va MC agentlarining konsentratsiyasi har xil bo'lgan A), B), E) va floresans spektroskopiyasi yordamida kuzatiladigan CspB. , C)  PEG8, B), D)  PEG1 yoki E), F) �x20. Eksperimental ma'lumotlar ramzlar sifatida ko'rsatiladi, ikkinchisining katlama modeliga mos keladigan global ma'lumotlar to'g'ri chiziqlar bilan ifodalanadi. Umumiy barqarorlik uchun mos qiymatlar (ΔG) va katlama kooperativligi (m)  S1 -jadvalda keltirilgan.

Keyinchalik, biz ochish uchun kuchliroq va qutbli denaturant guanidinium xloriddan (GdmCl) foydalandik. BsCspB kimyoviy jihatdan. PEG8 bilan ham, PEG1 bilan ham biz barqarorlikni topdik BsCspB karbamid bilan bir xil darajada (rasm   1 𠂜, D, jadval va#x02005S1 -rasm). Ajablanarlisi shundaki, PEGni Dex20 qutbli MC agenti bilan taqqoslaganda, biz aniq natijaga erishdik: o'tish nuqtasining ortishi (v M) 10 va#x02009% ni solishtirish bilan bir xil darajada (w/v) 10 va#x02009% bilan PEG8 (w/v) Dex20 in the cases both of urea‐ and of GdmCl‐induced denaturing (Figure  1 𠂞, F, Table S1).

So far we can summarize that the stabilizing effect of MC agents on chemical unfolding of BsCspB is only dependent on the volume fraction of the crowder in solution and neither on the size or the polarity of the crowding agent, nor on the charge of the denaturant, nor on the viscosity of the buffer. Mechanistically, this can be interpreted as a pure excluded volume effect, because the volume occupied by the crowding agent is the only shared factor that governs the increase in stability towards chemical unfolding. Boshqa so'zlar bilan aytganda, BsCspB acts as an ideal system in which both the less polar PEG and the polar Dex macromolecules can equally be considered inert crowding agents.

In a next step, thermal denaturation of BsCspB in the presence of MC was applied to evaluate whether the stabilization seen so far is only dependent on chemical unfolding or if it can be thermodynamically generalized by confirmation in an experimentally different setup. It should be noted that, in contrast with chemical unfolding, the analysis of thermal unfolding by use only of a single parameter is not possible. The change in thermodynamic stability is reflected neither solely in the temperature midpoint (T m), nor exclusively in the change in enthalpy (ΔH), nor uniquely in the difference in the heat capacity (ΔC p), but might result from changes in any one, two, or all of these three parameters (see discussion in the Supporting Information). Thermal unfolding transitions for BsCspB as monitored by circular dichroism (CD) spectroscopy in the absence and in the presence of 10, 20, and 30 % (w/v) PEG8 (Figure  2 𠂚, Table S3) or Dex20 (Figure  2 𠂛, Table S3) show a pronounced increase in T m in the case of Dex20 but only an extenuated one in that of PEG8. In contrast, ΔH shows a strong increase with rising PEG8 concentration whereas increasing Dex20 concentration does not affect ΔH significantly (Figure  2 𠂜, Table S3). These divergent effects of MC agents seen for the thermal denaturation of BsCspB have also been noted previously in the cases of, for example, ubiquitin13 and lysozyme.14

Thermally induced unfolding of BsCspB as monitored by CD spectroscopy in the presence of dilute ( • ), 10 ( ○ ), 20 ( ▪ ), or 30 % (w/v) ( □ ) A) PEG8, or B)�x20. Experimental data are shown as symbols whereas data fitting to a two‐state folding model are represented by straight lines. Fitting values for the change in enthalpy (ΔH) and the midpoint of the transition (T m) are given in Table S3. C) The values of ΔH va T m depend on the amount of urea added to BsCspB present at dilute, 20 % (w/v) PEG8, or 20 % (w/v) Dex20 conditions (Figure S2 and Table S3). The inset refers to the fitted data obtained in (A) ( ▪ ) and (B) ( ▴ ). D) The overall stability of BsCspB as determined by fluorescence (lines) agrees very well with data obtained by using circular dichroism spectroscopy (symbols).

We additionally monitored the thermal denaturation of partially denatured samples of BsCspB by using 1, 2, or 3  m urea in combination with 0 or 20 % (w/v) PEG8 or 20 % (w/v) Dex20, thus being able to contrast the changes in free energy (ΔG) as observed for chemically induced unfolding with those in the thermally induced case (Figures  2 𠂜, D and S2, Table S3). Remarkably, this analysis illuminates a general stabilization effect of BsCspB due to addition of MC agents regardless of chemical or thermal unfolding or rather of the technique employed. So far we can conclude that, regardless of the size and polarity of the MC agent added to the solution, solely the weight volume fraction determines the increase in ΔG value of BsCspB (Figure  2 𠂝).

What molecular mechanism causes this stabilization? We turned to high‐resolution NMR spectroscopy to unravel potential local sites in BsCspB causing the overall stabilization seen by both fluorescence and CD spectroscopy. Firstly, thermal denaturation of BsCspB (Figure S3𠂚) as probed by one𠄍imensional proton NMR spectroscopy in the presence of various amounts of PEG1 or PEG35 yielded results similar to those seen with PEG8 (Figure S3𠂛𠄽, Table S4), thus confirming the analysis performed with CD spectroscopy. This agreement was further verified by using Dex20 as MC agent for thermal denaturation of BsCspB (Figure S3𠂞, Table S4). Moving on, the stepwise addition of PEG1, PEG8, PEG35, or Dex20 to 15 N‐labeled BsCspB (Figure S4𠂚) induced moderate chemical shift perturbations (CSPs) in two𠄍imensional 1 H, 15 N HSQC spectra (Figure S4𠂛𠄾). It is notable that neither the amplitudes nor the sequence�pendent courses of these changes match when PEG is compared with Dex20 (Figure S4𠂟–I). Moreover, comparison between residues experiencing CSPs significant larger than the mean as induced by PEG8 or Dex20 does not provide a general pattern even if the inherent properties of single amino acid residues are taken into account (Figure S4 J–M). We conclude that the structural characteristics of native BsCspB are highly conserved even when PEG or Dex20 is present.

What contributions, then, lead to the gain in stability seen for the native state of BsCspB if MC is present? We examined the hydrogen𠄋onding> network by assessment of temperature coefficients (TCs),15 finding a small number of subtle changes in the general pattern of the network (Figure S5𠂚𠄽). Note that the local hydrogen𠄋onding network present in BsCspB is nicely reproduced by using this method (Figure S5𠂞, F). Again, however, detailed analysis of the TCs’ dependence on amounts of MC agent added also does not give a general explanation for the monotonic increase in ΔG as presented in Figures  1 and ​ and2 2 𠂝.

Additionally, we determined the exchange of the amide protons (NHs) with solvent water protons by NMR spectroscopy with use of a modified Mexico16 sequence to characterize the dynamic features of the NHs contained in BsCspB on a millisecond timescale (Figure S6𠂚𠄿). On comparing the exchange rate constants (k masalan) in dilute solution with those in crowded solution we observed three effects (Figure  3 𠂚𠄼). Firstly, an increasing concentration of MC agents equalizes the mean of all k masalan values observed in the following manner: k dilute ex =(3.2ଘ.5) s 𢄡 , k PEG 8 k ex =(2.5ଔ.8) s 𢄡 , k Dex 20 , 12 wv ex =(1.8ଔ.1) s 𢄡 , and k Dex 20 , 24 wv ex =(1.5଒.4) s 𢄡 . Secondly, a decrease in the mean of k masalan values in the presence of increasing concentrations of MC agents is seen. Thirdly, a pronounced decrease in k masalan values of residues present in less protected loop regions and a parallel soft increase in k masalan values of residues present in β‐sheet positions, which were well protected in dilute conditions (Figure  3 𠂚), is apparent. This holds for Dex20 more than for PEG8, independently of the increase in viscosity (Table S2) but increasing with volume percentage of crowder in solution.

A) The accessibility of the amide protons in BsCspB depends on the amounts of MC agent added. The exchange rate constants (k masalan) were determined in the absence ( • ) and in the presence of 15 % (w/v) PEG8 ( • ), 12 % (w/v) Dex20 ( • ), or 24 % (w/v) Dex20 ( • ) by use of a modified Mexico sequence (cf. Figure S6). Residues comprising β‐sheets are indicated by use of a background colored in light gray. B), C) BsCspB (PDB ID: 1NMG) has been colored according to decreases (blue) or increases (orange) in exchange rate constants upon addition of 12 % (w/v) Dex20, relative to dilute conditions. Dark blue: Δk masalan<𢄠.1 s 𢄡 . Light blue: 𢄠.1<Δk masalanπ s 𢄡 . Light orange: 0<Δk masalanπ.1 s 𢄡 . Dark orange: Δk masalanϠ.1 s 𢄡 . Gray: n.a.

Two key conclusions can be derived from this. First of all, crowder polarity might play a crucial role in defining the protection effect against exchange of NHs with solvent protons. Thus, the more polar crowder Dex induced stronger protection than the less polar PEG8, which points to the important difference in electrostatic interactions offered by these two different MC agents. This correlates well with the difference between PEG and Dex20 seen in thermal (Figures  2 , S2, and S3, Tables S3 and S4) but not in chemical unfolding (Figure  1 , Table S1). Most likely, in chemical unfolding this difference in electrostatics between the polyether PEG and the polyalcohol Dex is covered by the presence of the strongly polar co‐solute urea and even more so by increasing ionic strength in the presence of GdmCl.17

Secondly, the increase in protection as observed increases monotonously with crowder concentration. Because very mobile NHs are readily prone to exchange with water protons, due to the absence of a hydrogen bond (Figure S6 G, H), the increased protection of those flexible and highly mobile regions points to a decrease in mobility. In other words, more rigid loop regions contribute to increased thermodynamic stability of the native state of BsCspB if MC is present. This increase in loop rigidity contributing to the native state stability is more pronounced in the case of Dex than in that of PEG, presumably due to electrostatic interactions. Notably, both MC agents are able to induce qualitatively a similar trend towards stabilization. The β‐sheet regions of BsCspB show only small changes in exchange rate constants of NHs upon addition of MC agents.

In this study we have applied a systematic approach to understand the impact of MC on the stability of a native protein at atomic resolution in a convergent manner. We have precisely investigated the chemically and thermally induced unfolding of BsCspB by use of fluorescence, CD, and NMR spectroscopic experiments under a multiplicity of different MC conditions. A comprehensive thermodynamic analysis of the acquired data has allowed us to come to a systematic conclusion on the MC effect on BsCspB: we find it to depend solely on the weight per volume fraction added, and not on the type of macromolecule used to mimic MC nor on its size. We can thus show that the thermodynamic stability of BsCspB can be specifically adjusted through addition of a macromolecular compound, regardless of its properties, merely and simply through the macromolecular presence at concentrations similar to those in vivo. High‐resolution NMR spectroscopy has illuminated the fact that MC agents significantly reduce the exchange of mobile NHs that are not involved in hydrogen bonding with solvent protons𠅊gain, independently of the type of MC agent but in a manner dependent on the weight per volume fraction added. We conclude that this increase in rigidity acts as a driving force for the thermodynamic stabilization of BsCspB if MC is present. This stabilization arises from a modified accessibility of NHs to the solvent, thus resulting in a different microenvironment in that presence of MC from that in the dilute scenario. We speculate that this change in solvent accessibility unraveled here in the case of BsCspB holds as a general explanation for the MC effect affecting the thermodynamic stability of proteins.

Manfaatlar to'qnashuvi

The authors declare no conflict of interest.


MATERIALLAR

Reaktivlar

Proteins to be analyzed, dissolved in an appropriate buffer

All of the following reagents are available from SigmaAldrich. http://www.sigmaaldrich.com

Guanidine-HCl (step 1A and 1B ) [SIGMA # G7153] (see Reagent setup)

Sodium hydroxide pellets (Steps 1A, 1B and 1C) [reagent grade Sigma-Aldrich # 22146] (see Reagent setup)

Sodium carbonate (Step 1C) [Sigma-Aldrich # 22232]

Sodium citrate (Step 1C) [Sigma #S1804 ]

Bovine Serum Albumin Cohn Fraction V (BSA) - (Step 1C) [Sigma A3059] (see Reagent setup)

Copper sulphate (Step 1C) [Sigma C1297]

Uskunalar

Circular dichroism instrument (see Equipment setup)

Microtiter plate reader (step 1C)

Microtiter plates (step 1C)

0.1 to 0.2 micron filters (Millipore corporation http://www.millipore.com)

Reagent Setup - Timeline 30 minutes to 2 hours

!CAUTION Solutions of Guanidine-HCl, sodium hydroxide and Benedict’s reagent are caustic. Wear gloves.

6M Guanidine-HCl, pH 7.1 (step 1A)

Dissolve 57.3 mg guanidine-HCl in approximately 90 ml water. Adjust the pH to 7.1 with either 1 M HCl or by adding solid NaOH pellets. Adjust the volume to 100 ml. Keeps indefinitely in a glass bottle with a plastic cap.

6M Guanidine-HCl, pH 12.5 (step 1A)

Dissolve 57.3 mg guanidine-HCl in approximately 90 ml of water. Adjust the pH to 12.5 by adding NaOH pellets. Adjust the volume to 100 ml. This solution should be checked before use and the pH adjusted if necessary to > 12 by adding solid NaOH pellets, as carbon dioxide absorbed from the air will lower the pH.

6M Guanidine-HCl, pH 6.5 (Step 1B)

Dissolve 57.3 mg guanidine-HCl in approximately 90 ml water. Adjust the pH to 6.5 with either 1 M HCl or by adding solid NaOH pellets. Adjust the volume to 100 ml. Keeps indefinitely in a bottle with a plastic cap.

Benedicts Reagent (step 1C)

Combine 50 grams of sodium carbonate with 86.5 gm of sodium citrate in 300 ml of water. Dissolve by stirring on hot plate. Filter though Whatman # 1 filter paper. Add 8.63 gm of CuSO4 dissolved in 50 ml of H2O. Dilute to 500 ml. This solution lasts about 1 year in a brown bottle at room temperature.

3% Sodium hydroxide (NaOH)

Dissolve 3 gm of sodium hydroxide pellets in 100 ml of water.

1 mg/ml BSA

Dissolve 2 mg of bovine serum albumin in 1 ml of water and filter through a Millipore filter. The concentration of BSA in mg/ml = A280/0.68. Dilute to a final concentration of 1 mg/ml.

Uskunani sozlash

CD MACHINES

For data collection from approximately 700 to 175 nm, machines can be obtained from Applied Photophysics Ltd (http://www.photophysics.com) Aviv Biomedical (http://www.avivbiomedical.com/) Jasco Inc. (http://www.jascoinc.com/) or Olis (http://www.olisweb.com/products/cd/). For data collection at lower wavelengths there are CD machines than use beam line radiation from synchrotrons: the NSLS Brookhaven, USA, (beam lines U9b and Ul1) ISA in Aarhus, Denmark (UV_1) the SRS Daresbury, UK (CD12) HSRC/HiSOR, Hiroshima, Japan BESSY2 in Berlin, Germany the BSRF in Beijing, China and the NSRL in Hefei, China.

CRITICAL STEP

CD machines must be calibrated on a regular basis to check that the ellipticity values and wavelengths are correct 52 , 57 . A commonly used calibration standard, crystallized CSA, (1S)-(+)-Camphor-10-sulfonic acid (Aldrich, C2107 http://www.sigmaaldrich.com) 1 mg/ml in a 1 cm cell, has an absorbance at 285 nm of 0.1486 and an ellipticity band with a peak at 291 nm of 335 millidegrees. In addition, the ratio of ellipticity of camphor-10-sulfonic acid at 192.5 to 290.5 should be between 2.05 and 2.08. These numbers correspond to a Δε of 2.36 at 290.5 nm and 4.90 at 192.5 nm 17 , 30 .

CLEANING CUVETTES- timeline 10 minutes to overnight

Cuvettes for CD measurements must be clean and dry. Quartz cells can be cleaned by soaking in: mild detergent solutions available from several cell manufacturers, such as Hellma a mixture of 30% concentrated HCl and 70% ethanol or concentrated nitric acid. Protein residues can be dissolved using 6M guanidine-HCl. After soaking, cells should be rinsed with water and ethanol and either dried by suction using an aspirator or blown dry with nitrogen or compressed air that has passed through a filter to remove impurities. If residual proteins are not removed by the previous cleaning agents, filling the cells with Chromerge (a mixture of potassium chromate in concentrated sulfuric acid) (Fisher scientific C577-12 https://www1.fishersci.com) and immediately rinsing out with water and drying usually is effective. This method is the best method for cleaning cells with 0.01 or 0.02 cm path lengths.

CAUTION Nitric acid, HCl and Chromerge are very caustic and will burn holes in lab coats and damage clothing. Wear gloves.

STARTING THE CD MACHINE – timeline 30 minutes

CD machines have very powerful lamps that promote the ionization of oxygen to ozone. Ozone is toxic and also will quickly destroy the mirrors in the optics of the machines. Most CD machines must be flushed with nitrogen to remove oxygen before the machine is turned on and during operation. Nitrogen sources include tanks of pre-purified nitrogen, which last about 5 hours and high-pressure liquid nitrogen tanks, which produce gas, and will last one to two weeks depending on usage. Commercial nitrogen should be free of oxygen and most other impurities, but some manufacturers suggest using a trap to remove impurities to be on the safe side. If you are new to CD and there is no one to teach you the operation of your specific machine call the manufacturer and ask for their start up protocol.


Qo'shimcha

Enthalpy, entropy convergence for hydrophobic hydration

In his massive and influential 1979 review of protein stability [112], one of the observations that Privalov made was that at about 100–110 °C, compact globular proteins converged on the same value of specific enthalpy of unfolding (cal/g). He found a similar convergence for the specific entropy of unfolding around 110 °C, although the effect was not nearly as clear.

In 1986, Robert Baldwin published a keystone paper [116] in which he compared the thermodynamics of liquid hydrocarbon neat transfer to the unfolding of the enzyme lysozyme. He found that for six hydrocarbons (benzene, toluene, ethylbenzene, cyclohexane, n-pentane, and n-hexane), TH va TS were approximately the same: TH = 22 ± 5 °C, and TS = 113 ± 3 °C. Footnote 7 Baldwin then applied these values to the temperature-dependent unfolding of hen lysozyme by splitting protein stability into a hydrophobic effect (HE) and a non-hydrophobic effect (NHE, ∆Xobs = ∆XHE + ∆XNHE), and assuming that (1) ∆C°P.(U) is entirely due to the HE, and (2) for the effect of hydrophobicity on protein unfolding, TH va TS are identical to the liquid hydrocarbon neat transfer values. Using Eqs. (4) and (6) (main text), Baldwin calculated HE ∆H°298 and ∆S°298, and then by subtracting from the net observed ∆H°298 and ∆S°298, he calculated the NHE values. Both NHE values were temperature-independent: ∆H°298(U, NHE) = + 217.8 ± 0.7 kJ/mol, and ∆S°298(U, NHE) = + 2294 ± 3 J/K/mol. The former value was interpreted as the cost, upon unfolding, of breaking polar interactions (e.g., H-bonds), and the latter the increase in conformational entropy, i.e., the release of polypeptide chain positioning constraints.

In 1990, Murphy and Gill took this analysis a step further, defining the notion of “convergence” temperatures for enthalpy and entropy [10, 13, 114, 115]. They started by applying Baldwin’s HE/NHE breakdown to the temperature dependence of enthalpy, considering a series of homologous compounds (e.g., noble gases, alkanes, alcohols, amines) which shared the same functional group and differed only in their hydrophobicity (e.g., size of noble gas or number of carbons). The hydration enthalpy can then be envisioned as comprising a hydrophobic term that increases with solute hydrophobicity, and a non-hydrophobic term that is independent of hydrophobicity. Enthalpy convergence would then occur at some temperature, TH,conv, at which all of the homologous compounds in the series have the same hydration enthalpy, ∆Hconv. Thus, at the convergence temperature, ∆H° of hydration depends only on the nature of the series (i.e., the functional group), and not on the individual compound. With this in mind, the temperature dependence of ∆H (main text Eq. 2), can be cast as

Normally, one would use Eq. (19) along with measured values of ∆H° at various temperatures to determine ∆C°P.. If, however, one already knows ∆C°P. for each compound in the series, then measured values of ∆H° at a specific reference temperature (e.g., 298 K) can be used in the following version of Eq. (19):

Thus, a plot of ∆H°298 vs. ∆C°P. for each compound in the series will give a straight line with slope = (298 − Tconv) and intercept = ∆H°(Tconv). This is sometimes referred to as an MPG (Murphy–Privalov–Gill) plot. Because ∆H°(Tconv) characterizes the entire series, it must apply to what remains the same in the series, i.e., the non-hydrophobic component of the observed net ∆H° thus, it would be different for a series of alkanes vs. alcohols vs. amines. Boshqa tarafdan, Tconv should be the same for alkanes, alcohols, and amines because they all differ by the same hydrophobic parameter, i.e., the number of carbons.

A similar analysis of entropy leads to this adaptation of Eq. (3) in the main text:

The MPG plot here would be ∆S°298 vs. ∆C°P., with slope = ln(298/TS,conv), and intercept = ∆S°(TS,conv). As with enthalpy, ∆S°(Tconv) characterizes the entire series, so it must apply to the non-hydrophobic component of the observed net ∆S°.

For example, MPG plots of Baldwin’s liquid hydrocarbon neat transfer data [116] do show a linear correlation (Fig. 11), although the relationship is not very strong for ∆H (R 2 = 0.6). From the slopes, the convergence temperatures are calculated to be TS,conv = 106 ± 5 °C TH,conv = 40 ± 6 °C. Note that these temperatures are close to, but not identical to TS va TH.

MPG plots of liquid hydrocarbon neat transfer. a Entropy b enthalpy liquids are benzene, toluene, ethylbenzene, cyclohexane, n-pentane, and n-hexane

Figure 11a, with its excellent linearity, suggests that entropy convergence for the neat transfer of this series of liquid hydrocarbons does occur. The relative uncertainty in the convergence temperature is good, at only 4% (= 100 × 4.7/106). (For ∆S°(TS,conv), however, it is 70%!) Thus, we might expect to see a fairly robust isosbestic point in the ∆S° vs. T plot for neat transfer (Fig. 12). Sadly, that is not actually the case.

Temperature dependence of the entropy of neat transfer of liquid hydrocarbons

The first thing to note from Fig. 12 is that for all six liquids, TS, the temperature at which each curve crosses the T-axis, lies between 382 and 390 K this matches the average value reported above (113 ± 3 °C). Most of the curves cross each other (shared values of ∆S°) between about 360 and 390 K this also matches the calculated convergence temperature from the MPG plot, 106 ± 5 °C. However, there are several intersections below 360 K, and several more above 400 K. The lack of a clear isosbestic point suggests that the entropic convergence behavior is not nearly as robust as one might have expected from the MPG plot. A similar “smeared” isosbestic is observed in the ∆H° vs. T curve intersections, stretching from 300 to 345 K (data not shown). At least some of this “blurring” of the expected isosbestic point has been shown to be due to the temperature dependence of ∆CP. [117]. Beyond this though, one may question the assumptions in the convergence derivation, namely, that the hydrophobic and non-hydrophobic contributions can be separated out and that the former really are identical for every member of the homologous series of compounds. Graziano [32] and Pratt [117] have supported the existence of entropy convergence for families of small solutes, but rejected enthalpy convergence (Graziano, personal communication).

Murphy, Privalov, and Gill published an extremely influential paper in 1990 in which they compared MPG plots of Baldwin’s liquid hydrocarbon data with nonpolar gas hydration, solid → water transfer of nonpolar cyclic peptides, and protein unfolding [10]. They found that the slopes of all four of the ∆S° vs. ∆C°P. plots were nearly identical, ranging from − 0.23 ± 0.04 to − 0.28 ± 0.01. Thus TS,conv for all four of these sets of compounds lie in a fairly narrow range between 102 ± 15 and 121 ± 6 °C this pointed to the “dominant role that water [and the hydrophobic effect] play in determining the [entropy] of hydration of [all of] these compounds” [10].

Murphy, Privalov, and Gill drew another interesting conclusion from the intercepts of their MPG plots [10]. They determined ∆S°(TS,conv) to be − 78.5 ± 2.5 J/K/mol for nonpolar gas hydration vs. − 6 ± 4 J/K/mol for liquid neat transfer this is expected, because gases lose much more freedom of motion upon transfer to the aqueous phase than liquids do. Meanwhile, ∆S°(TS,conv) was quite similar for protein unfolding (18 ± 1 J/K/mol) and the transfer of solid nonpolar cyclic peptides into water (16 ± 1 J/K/mol). This corroborated previous findings that in terms of packing and freedom of motion, the protein interior is best modeled as a solid organic compound, rather than a liquid [118,119,120].

To give some idea of the linearity of the MPG plots in the 1990 Ilm paper, Fig. 13 plots the nonpolar gas hydration and protein unfolding results. Note that once again, the linear fits are good (R 2 = 0.89 for gas hydration, 0.96 for protein unfolding), and the slopes are fairly close (− 0.068 and − 0.077). In light of how influential the 1990 Murphy, Privalov, and Gill paper was, it is surprising that no one seems to have checked to see whether these gases and proteins actually demonstrated isosbestic behavior in their ∆H° vs. T plots. In Fig. 14 I have plotted the five members of each series that lie closest to the linear fit line in the MPG plots. As with liquid hydrocarbon neat transfer (Fig. 12), many of the crossings lie in a fairly narrow range of temperature (355–365 K for A and 370–380 K for B), but a number of crossings occur far outside this range.

MPG enthalpy plots of a nonpolar gas hydration and b protein unfolding data from ref. [101]. Without giving any reason, MPG omitted the parvalbumin unfolding data point (red) from their published plot

Temperature dependence of the enthalpy of a nonpolar gas hydration, and b protein unfolding

Perhaps the most interesting part of this story about convergence came to light in the years after 1990, as more protein unfolding thermodynamic data became available. Twelve proteins were originally tabulated in Privalov and Gill’s 1988 review [101], 11 of which were included in their 1990 MPG plot [10]. It turned out that not only was the omitted point (suspiciously?) far off the fit line (Fig. 14b), but the 11 points included were extraordinarily unusual in their linearity.

This was discovered in 1997, when Robertson and Murphy published a review in which they tabulated unfolding thermodynamic data for 65 globular proteins of known structure [102]. The MPG plots in this paper had R 2 values of only 0.36 and 0.33 unsurprisingly, the convergence temperatures calculated from the slopes of these plots (66 °C for enthalpy, 65 °C for entropy) were much different from those in the 1990 paper (102 °C for enthalpy, 106 °C for entropy). Footnote 8 Robertson and Murphy’s conclusion from these disappointing MPG plots was that the convergence behavior for this much larger set of unfolded proteins was “not very compelling.” Huang and Chandler [121], using Lum–Chandler–Weeks theory [18], showed that the convergence temperature should decrease with increasing protein size, thus “one may not expect to observe a convergence temperature for the entropy of unfolding for all proteins.” Considering the diversity of protein groups that are exposed to water upon unfolding, as well as the change in shape, it is not surprising that proteins do not behave like a set of homologous compounds all with the same functional group. (And as we have seen, the convergence behavior even in this best of cases is not as clear as one might hope.) This failure to observe convergence behavior in the larger data sets has been recognized by some authors [20], but has been ignored by others [99].

It is important to conclude this discussion by stressing that enthalpy and entropy convergence do not generally exist for protein unfolding, and the existence of enthalpy convergence for hydrophobic hydration can be questioned as well.

Entropy is a measure of freedom of motion

One way to measure entropy is given by Boltzmann’s entropy equation,

qayerda R is the gas law constant, 8.314 J/K/mol, and V is the number of ways that a system can be arranged. For an ideal gas, V can be a measure of positions within a volume of space available to the particle. Excluding the particle from a portion of the volume will lower V and thus decrease entropy.

We can demonstrate this by combining the first and second laws of thermodynamics to get, for a fully reversible reaction,

Furthermore, since dU = CV·dT, Eq. (23) becomes

Using the ideal gas law (PV = nRTP/T = R/ (overline) ), we get

For an isothermal process, dT = 0, so

For the reversible isothermal compression of an ideal gas, dV is negative and thus dS would be negative as well. In other words, restricting the available space curtails freedom of motion, which in turn lowers entropy.


Videoni tomosha qiling: OQSILLAR BIOSINTEZI (Yanvar 2022).