vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RASvavilov-57Research ArticleСтатьиArticlesИССЛЕДОВАНИЕ ТЕРМОСТАБИЛЬНОСТИ МУТАНТНЫХ ФОРМ БЕЛКА БАРНАЗЫ С ИСПОЛЬЗОВАНИЕМ ПРОГРАММНОГО КОМПЛЕКСА MOLKERNSTUDY OF THE THERMAL STABILITY OF BARNASE PROTEIN MUTANTS WITH MOLKERN SOFTWAREФоминЭ. С.FominE. S.fomin@bionet.nsc.ruАлемасовН. А.AlemasovN. A.fomin@bionet.nsc.ruФедеральное государственное бюджетное научное учреждение «Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наук», Новосибирск, РоссияРоссияInstitute of Cytology and Genetics, SB RAS, Novosibirsk, RussiaRussian Federation201213122014162415426Copyright © Фомин Э.С., Алемасов Н.А., 20142014Фомин Э.С., Алемасов Н.А.Fomin E.S., Alemasov N.A.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/57

Выполнено исследование термостабильности ряда мутантных форм белка барназы методом λ-динамики. Метод реализован в рамках программного комплекса MOLKERN. Для исследования выбраны мутации, включающие аминокислоты с ненулевым зарядом, точность расчетов которых в методе λ-динамики почти в 1∕4 случаев существенно отличается от экспериментальных данных (> 10 кДж/моль). Предложены оригинальные модификации метода λ-динамики, связанные с построением λ-потенциалов и учетом эффектов изменения заряда. Результаты выполненных расчетов (для мутации R72G) показали лучшее согласие с экспериментальными значениями, чем результаты других авторов.

A thermal stability study of several barnase mutants has been carried out by the λ dynamics method. The method has been implemented in the MOLKERN software package. Mutations of amino acids with non-zero charge are chosen for the study, because in this case λ dynamics gives results differing dramatically (> 10 kJ/mol) from experimental data in nearly one-fourth of cases. A new modification of λ potentials is proposed, which takes into account charge changes, as well as the Net-Q method, in order to obtain reliable charge distributions for charged amino acids. The results obtained for the R72G mutation show a better agreement with experimental values than the results of other authors.

барназамолекулярная динамикаλ-динамикаразность свободных энергийтермостабильность белковbarnasemolecular dynamicsλ dynamicsfree energy differenceprotein thermal stabilityReferencesСеров А.Е., Одинцева Е.Р., Упоров И.В., Тишков В.И. Использование карты Рамачандрана для повышения термостабильности бактериальной формиатдегидрогеназы // Биохимия. 2005. Т. 70. Вып. 7. С. 974–979.Серов А.Е., Одинцева Е.Р., Упоров И.В., Тишков В.И. Использование карты Рамачандрана для повышения термостабильности бактериальной формиатдегидрогеназы // Биохимия. 2005. Т. 70. Вып. 7. С. 974–979.Серов А.Е., Тишков В.И. Роль остатков пролина в стабильности прокариотических и эукариотических формиатдегидрогенах // Вестник Моск. ун-та. Сер. 2. Химия. 2002. Т. 43. № 6. С. 345–349.Серов А.Е., Тишков В.И. Роль остатков пролина в стабильности прокариотических и эукариотических формиатдегидрогенах // Вестник Моск. ун-та. Сер. 2. Химия. 2002. Т. 43. № 6. С. 345–349.Полянский А.А., Косинский Ю.А., Ефремов Р.Г. Локальные температурные изменения подвижности в молекулах тиоредоксинов влияют на их термостабильные свойства // Биоорган. химия. 2004. Т. 30. № 5. С. 470–480.Полянский А.А., Косинский Ю.А., Ефремов Р.Г. Локальные температурные изменения подвижности в молекулах тиоредоксинов влияют на их термостабильные свойства // Биоорган. химия. 2004. Т. 30. № 5. С. 470–480.Фомин Э.C., Алемасов Н.А., Чирцов А.С., Фомин А.Э. Библиотека программных компонент MOLKERN для построения программ молекулярного моделирования // Биофизика. 2006. Т. 51. Вып. 7. С. 110–113.Фомин Э.C., Алемасов Н.А., Чирцов А.С., Фомин А.Э. Библиотека программных компонент MOLKERN для построения программ молекулярного моделирования // Биофизика. 2006. Т. 51. Вып. 7. С. 110–113.Bartels C., Schaefer M., Karplus M. Determination of equilibrium properties of biomolecular systems using multidimensional adaptive umbrella sampling // J. Chem. Phys. 1999. V. 111. No. 17. P. 8048–8068.Bartels C., Schaefer M., Karplus M. Determination of equilibrium properties of biomolecular systems using multidimensional adaptive umbrella sampling // J. Chem. Phys. 1999. V. 111. No. 17. P. 8048–8068.Bash P.A., Singh U.C., Brown F.K. Calculation of the relative change in binding free energy of a protein inhibitor complex // Science. 1987. V. 235. No. 4788. P. 574–576.Bash P.A., Singh U.C., Brown F.K. Calculation of the relative change in binding free energy of a protein inhibitor complex // Science. 1987. V. 235. No. 4788. P. 574–576.Bayly C.I., Cieplak P., Cornell W.D., Kollman P.A. A well-behaved electrostatic potential based method using charge restraints for determining atom-centered charges: The RESP model // J. Phys. Chem. 1993. V. 97. P. 10269–10280.Bayly C.I., Cieplak P., Cornell W.D., Kollman P.A. A well-behaved electrostatic potential based method using charge restraints for determining atom-centered charges: The RESP model // J. Phys. Chem. 1993. V. 97. P. 10269–10280.Bennett C.H. Efficient estimation of free energy differences from Monte Carlo data // J. Comput. Phys. 1976. V. 22. No. 2. P. 245–268.Bennett C.H. Efficient estimation of free energy differences from Monte Carlo data // J. Comput. Phys. 1976. V. 22. No. 2. P. 245–268.Berendsen H.J.C., Postma J.P.M., DiNola A., Haak J.R. Molecular dynamics with coupling to an external bath // J. Chem. Phys. 1984. V. 81. P. 3684–3690.Berendsen H.J.C., Postma J.P.M., DiNola A., Haak J.R. Molecular dynamics with coupling to an external bath // J. Chem. Phys. 1984. V. 81. P. 3684–3690.Beutlera T.C., Marka A.E., van Schaik R.C. et al. Avoiding singularities and numerical instabilities in free energy calculations based on molecular simulations // Chem. Phys. Lett. 1994. V. 222. No. 6. P. 529–539.Beutlera T.C., Marka A.E., van Schaik R.C. et al. Avoiding singularities and numerical instabilities in free energy calculations based on molecular simulations // Chem. Phys. Lett. 1994. V. 222. No. 6. P. 529–539.Boczko E.M., Brooks C.L. First-principles calculation of the folding free energy of a three-helix bundle protein // Science. 1995. V. 269. No. 5222. P. 393–396.Boczko E.M., Brooks C.L. First-principles calculation of the folding free energy of a three-helix bundle protein // Science. 1995. V. 269. No. 5222. P. 393–396.Buckle A.M., Henrick K., Fersht A.R. Crystal structural analysis of mutations in the hydrophobic cores of barnase // J. Mol. Biol. 1993. V. 234. No. 3. P. 847–860.Buckle A.M., Henrick K., Fersht A.R. Crystal structural analysis of mutations in the hydrophobic cores of barnase // J. Mol. Biol. 1993. V. 234. No. 3. P. 847–860.Bycroft M., Matouschek A., Kellis J.T. Jr. et al. Detection and characterization of a folding intermediate in barnase by NMR // Nature. 1990. V. 346. No. 6283. P. 488-490.Bycroft M., Matouschek A., Kellis J.T. Jr. et al. Detection and characterization of a folding intermediate in barnase by NMR // Nature. 1990. V. 346. No. 6283. P. 488-490.Chen B., Potoff J.J., Siepmann J.I. Adiabatic nuclear and electronic sampling Monte Carlo simulations in the gibbs ensemble: Application to polarizable force fields for water // J. Phys. Chem. B. 2000. V. 104. P. 2378–2390.Chen B., Potoff J.J., Siepmann J.I. Adiabatic nuclear and electronic sampling Monte Carlo simulations in the gibbs ensemble: Application to polarizable force fields for water // J. Phys. Chem. B. 2000. V. 104. P. 2378–2390.Chen J., Martinez T.J. QTPIE: Charge transfer with polarization current equalization. A fluctuation charge model with correct asymptotics // Chem. Phys. Lett. 2007. V. 463. P. 315–320.Chen J., Martinez T.J. QTPIE: Charge transfer with polarization current equalization. A fluctuation charge model with correct asymptotics // Chem. Phys. Lett. 2007. V. 463. P. 315–320.Clarke J., Fersht A.R. Engineered disulfide bonds as probes of the folding pathway of barnase: Increasing the stability of proteins against the rate of denaturation // Biochemistry. 1993. V. 32. No. 16. P. 4322–4329.Clarke J., Fersht A.R. Engineered disulfide bonds as probes of the folding pathway of barnase: Increasing the stability of proteins against the rate of denaturation // Biochemistry. 1993. V. 32. No. 16. P. 4322–4329.Dang L.X., Merz K.M. Jr., Kollman P.A. Free energy calculations on protein stability: Thr-157 → Val-157 mutation of T4 lysozyme // J. Amer. Chem.Soc. 1989. V. 111. No. 22. P. 8505–8508.Dang L.X., Merz K.M. Jr., Kollman P.A. Free energy calculations on protein stability: Thr-157 → Val-157 mutation of T4 lysozyme // J. Amer. Chem.Soc. 1989. V. 111. No. 22. P. 8505–8508.Fersht A.R., Daggett V. Protein folding and unfolding at atomic resolution // Cell. 2002. V. 108. No. 4. P. 573–582.Fersht A.R., Daggett V. Protein folding and unfolding at atomic resolution // Cell. 2002. V. 108. No. 4. P. 573–582.Fomin E.S. Consideration of data load time on modern processors for the verlet table and linked cell algorithms // J. Comput. Chem. 2011. V. 32. No. 7. P. 1386–1399.Fomin E.S. Consideration of data load time on modern processors for the verlet table and linked cell algorithms // J. Comput. Chem. 2011. V. 32. No. 7. P. 1386–1399.Gilisa D., Rooman M. Stability changes upon mutation of solvent-accessible residues in proteins evaluated by database-derived potentials // J. Mol. Biol. 1996. V. 257. No. 5. P. 1112–1126.Gilisa D., Rooman M. Stability changes upon mutation of solvent-accessible residues in proteins evaluated by database-derived potentials // J. Mol. Biol. 1996. V. 257. No. 5. P. 1112–1126.Hartley R.W. Barnase and barstar: two small proteins to fold and fit together // Trends Biochem. Sci. 1989. V. 14. No. 11. P. 450–454.Hartley R.W. Barnase and barstar: two small proteins to fold and fit together // Trends Biochem. Sci. 1989. V. 14. No. 11. P. 450–454.Horovitz A., Matthews J.M., Fersht A.R. a-Helix stability in proteins. II. Factors that influence stability at an internal position // J. Mol. Biol. 1992. V. 227. No. 2. P. 560–568.Horovitz A., Matthews J.M., Fersht A.R. a-Helix stability in proteins. II. Factors that influence stability at an internal position // J. Mol. Biol. 1992. V. 227. No. 2. P. 560–568.Johnson C.M., Fersht A.R. Protein stability as a function of denaturant concentration: The thermal stability of barnase in the presence of urea // Biochemistry. 1995. V. 34. No. 20. P. 6795–6804.Johnson C.M., Fersht A.R. Protein stability as a function of denaturant concentration: The thermal stability of barnase in the presence of urea // Biochemistry. 1995. V. 34. No. 20. P. 6795–6804.Kellis J.T. Jr., Nyberg K., Fersht A.R. Energetics of complementary side chain packing in a protein hydrophobic core // Biochemistry. 1989. V. 28. No. 11. P. 4914–4922.Kellis J.T. Jr., Nyberg K., Fersht A.R. Energetics of complementary side chain packing in a protein hydrophobic core // Biochemistry. 1989. V. 28. No. 11. P. 4914–4922.Kirkwood J.G. Statistical mechanics of fluid mixtures // J. Chem. Phys. 1935. V. 3/5. P. 300–314.Kirkwood J.G. Statistical mechanics of fluid mixtures // J. Chem. Phys. 1935. V. 3/5. P. 300–314.Knight J.L., Brooks C.L. III. Lambda-dynamics free energy simulation methods // J. Comput. Chem. 2009. V. 30. P. 1692–1700.Knight J.L., Brooks C.L. III. Lambda-dynamics free energy simulation methods // J. Comput. Chem. 2009. V. 30. P. 1692–1700.Kollman P. Free energy calculations: Applications to chemical and biochemical phenomena // Chem. Rev. 1993. V. 93. No. 7. P. 2395–2417.Kollman P. Free energy calculations: Applications to chemical and biochemical phenomena // Chem. Rev. 1993. V. 93. No. 7. P. 2395–2417.Kumar M.D.S., Bava K.A., Gromiha M.M et al. ProTherm and ProNIT: thermodynamic databases for proteins and protein-nucleic acid interactions // Nucl. Acids Res. 2006. V. 34 (Database issue). P. D204–D206.Kumar M.D.S., Bava K.A., Gromiha M.M et al. ProTherm and ProNIT: thermodynamic databases for proteins and protein-nucleic acid interactions // Nucl. Acids Res. 2006. V. 34 (Database issue). P. D204–D206.Lehmann M., Wyss M. Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution // Curr. Opin.Biotechnol. 2001. V. 12. P. 371–375.Lehmann M., Wyss M. Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution // Curr. Opin.Biotechnol. 2001. V. 12. P. 371–375.Matouschek A., Kellis J.T. Jr., Serrano L., Fersht A.R. Mapping the transition state and pathway of protein folding by protein engineering // Nature. 1989. V. 340. No. 6229. P. 122–126.Matouschek A., Kellis J.T. Jr., Serrano L., Fersht A.R. Mapping the transition state and pathway of protein folding by protein engineering // Nature. 1989. V. 340. No. 6229. P. 122–126.Martin C., Richard V., Salem M. et al. Refinement and structural analysis of barnase at 1.5 A resolution // Acta Crystallogr. D. Biol. Crystallogr. 1999. V. 55. No. 2. P. 386–398.Martin C., Richard V., Salem M. et al. Refinement and structural analysis of barnase at 1.5 A resolution // Acta Crystallogr. D. Biol. Crystallogr. 1999. V. 55. No. 2. P. 386–398.Meharenna Y.T., Poulos T.L. Using molecular dynamics to probe the structural basis for enhanced stability in thermal stable cytochromes P450 // Biochemistry. 2010. V. 49. No. 31. P. 6680–6686.Meharenna Y.T., Poulos T.L. Using molecular dynamics to probe the structural basis for enhanced stability in thermal stable cytochromes P450 // Biochemistry. 2010. V. 49. No. 31. P. 6680–6686.Messmer R.P. Cluster Model theory and its application to metal surface – adsorbate system // The Nature of the Surface Chemical Bond / Eds T.N. Rhodin, G. Ertl. Amsterdam, Noth-Holland Publ. Co., 1979. P. 51–111.Messmer R.P. Cluster Model theory and its application to metal surface – adsorbate system // The Nature of the Surface Chemical Bond / Eds T.N. Rhodin, G. Ertl. Amsterdam, Noth-Holland Publ. Co., 1979. P. 51–111.Morales J., Martinez T.J. Classical fluctuating charge theories: The maximum entropy valence bond method and relationships to previous models // J. Phys. Chem. 2001. V. 105A. P. 2842–2850.Morales J., Martinez T.J. Classical fluctuating charge theories: The maximum entropy valence bond method and relationships to previous models // J. Phys. Chem. 2001. V. 105A. P. 2842–2850.Mortier W.J., Ghosh S.K., Shankar S.J. Electronegativity-equalization method for the calculation of atomic charges in molecules // Am. Chem. Soc. 1986. V. 108. P. 4315–4320.Mortier W.J., Ghosh S.K., Shankar S.J. Electronegativity-equalization method for the calculation of atomic charges in molecules // Am. Chem. Soc. 1986. V. 108. P. 4315–4320.Oliferenko A.A., Palyulin V.A., Pisarev S.A. et al. Novel point charge models: reliable instruments for molecular electrostatics // J. Phys. Org. Chem. 2001. V. 14. P. 355–369.Oliferenko A.A., Palyulin V.A., Pisarev S.A. et al. Novel point charge models: reliable instruments for molecular electrostatics // J. Phys. Org. Chem. 2001. V. 14. P. 355–369.Pedersen J.S., Otzen D.E., Kristensen P. Directed evolution of barnase stability using proteolytic selection // J. Mol. Biol. 2002. V. 323. No. 1. P. 115–123.Pedersen J.S., Otzen D.E., Kristensen P. Directed evolution of barnase stability using proteolytic selection // J. Mol. Biol. 2002. V. 323. No. 1. P. 115–123.Pikkemaat M.G., Linssen A.B., Berendsen H.J., Janssen D.B. Molecular dynamics simulations as a tool for improving protein stability // Protein Eng. 2002. V. 15. No. 3. P. 185–192.Pikkemaat M.G., Linssen A.B., Berendsen H.J., Janssen D.B. Molecular dynamics simulations as a tool for improving protein stability // Protein Eng. 2002. V. 15. No. 3. P. 185–192.Prevost M., Wodak S.J., Tidor B., Karplus M. Contribution of the hydrophobic effect to protein stability: analysis based on simulations of the Ile-96-Ala mutation in barnase // Proc. Natl Acad. Sci. USA. 1991. V. 88. No. 23. P. 10880–10884.Prevost M., Wodak S.J., Tidor B., Karplus M. Contribution of the hydrophobic effect to protein stability: analysis based on simulations of the Ile-96-Ala mutation in barnase // Proc. Natl Acad. Sci. USA. 1991. V. 88. No. 23. P. 10880–10884.Puchkaev A.V., Koo L.S., de Montellano P.R.O. Aromatic stacking as a determinant of the thermal stability of CYP119 from Sulfolobus solfataricus // Arch. Biochem. Biophys. 2003. V. 409. No. 1. P. 52–58.Puchkaev A.V., Koo L.S., de Montellano P.R.O. Aromatic stacking as a determinant of the thermal stability of CYP119 from Sulfolobus solfataricus // Arch. Biochem. Biophys. 2003. V. 409. No. 1. P. 52–58.Rappe A.K., Goddard W.A. Charge equilibration for molecular dynamics simulations // J. Phys. Chem. 1991. V. 95. P. 3358–3363.Rappe A.K., Goddard W.A. Charge equilibration for molecular dynamics simulations // J. Phys. Chem. 1991. V. 95. P. 3358–3363.Rick S.W., Stuart S.J., Berne B.J. Dynamical fluctuating charge force fields: Application to liquid water // J. Chem. Phys. 1994. V. 101. P. 6141–6156.Rick S.W., Stuart S.J., Berne B.J. Dynamical fluctuating charge force fields: Application to liquid water // J. Chem. Phys. 1994. V. 101. P. 6141–6156.Rouxa B., Karplus M. Ion transport in a model gramicidin channel. Structure and thermodynamics // Biophys. J. 1991. V. 59. No. 5. P. 961–981.Rouxa B., Karplus M. Ion transport in a model gramicidin channel. Structure and thermodynamics // Biophys. J. 1991. V. 59. No. 5. P. 961–981.Sanderson R.T. An interpretation of bond lengths and classification of bonds // Science. 1951. V. 114. P. 670–672.Sanderson R.T. An interpretation of bond lengths and classification of bonds // Science. 1951. V. 114. P. 670–672.Seeliger D., de Groot B.L. Protein thermostability calculations using alchemical free energy simulations // Biophys. J. 2010. V. 98. No. 10. P. 2309–2316.Seeliger D., de Groot B.L. Protein thermostability calculations using alchemical free energy simulations // Biophys. J. 2010. V. 98. No. 10. P. 2309–2316.Serrano L., Kellis J.T. Jr., Cann P. et al. The folding of an enzyme: II. Substructure of barnase and the contribution of different interactions to protein stability // J. Mol. Biol. 1992. V. 224. No. 3. P. 783–804.Serrano L., Kellis J.T. Jr., Cann P. et al. The folding of an enzyme: II. Substructure of barnase and the contribution of different interactions to protein stability // J. Mol. Biol. 1992. V. 224. No. 3. P. 783–804.Simonson T., Archontis G., Karplus M. Free energy simulations come of age: Protein-ligand recognition // Accounts Chem. Res. 2002. V. 35. No. 6. P. 430–437.Simonson T., Archontis G., Karplus M. Free energy simulations come of age: Protein-ligand recognition // Accounts Chem. Res. 2002. V. 35. No. 6. P. 430–437.Tidor B., Karplus M. Simulation analysis of the stability mutant R96H of T4 lysozyme // Biochemistry. 1991. V. 30. No. 13. P. 3217–3228.Tidor B., Karplus M. Simulation analysis of the stability mutant R96H of T4 lysozyme // Biochemistry. 1991. V. 30. No. 13. P. 3217–3228.York D.M., Yang W. A chemical potential equalization method for molecular simulations // J. Chem. Phys. 1996. V. 104. P. 159–172.York D.M., Yang W. A chemical potential equalization method for molecular simulations // J. Chem. Phys. 1996. V. 104. P. 159–172.Zhu S.B., Singh S., Robinson G.W. A new flexible/polarizable water model // J. Chem. Phys. 1991. V. 95. P. 2791–2799.Zhu S.B., Singh S., Robinson G.W. A new flexible/polarizable water model // J. Chem. Phys. 1991. V. 95. P. 2791–2799.Zwanzig R.W. High-temperature equation of state by a perturbation method. I. Nonpolar gases // J. Chem. Phys. 1954. V. 22. No. 8. P. 1420–1427.Zwanzig R.W. High-temperature equation of state by a perturbation method. I. Nonpolar gases // J. Chem. Phys. 1954. V. 22. No. 8. P. 1420–1427.

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