Russian Journal of Biological Physics and Chemisrty Russian Journal of Biological Physics and Chemisrty АКТУАЛЬНЫЕ ВОПРОСЫ БИОЛОГИЧЕСКОЙ ФИЗИКИ И ХИМИИ 2499-9962 54315 Общая биофизика General biophysics Общая биофизика APPLICATION OF THE TIME-RESOLVED FLUORESCENCE TO THE CHARACTERIZATION OF PROTEINS UNFOLDING PATHWAYS ИСПОЛЬЗОВАНИЕ ВРЕМЯ-РАЗРЕШЕННОЙ ФЛУОРЕСЦЕНЦИИ БЕЛКОВ ДЛЯ ОПРЕДЕЛЕНИЯ СТАДИЙ ИХ ДЕНАТУРАЦИИ Немцева Е В Nemtseva E V enemtseva@sfu-kras.ru Герасимова М А Gerasimova M A Лащук О О Lashchuk O O Карузина Н Е Karuzina N E Гульнов Д В Gulnov D V Мельник Б С Melnik B S Сибирский федеральный университет; Институт биофизики СО РАН ФИЦ «Красноярский научный центр СО РАН» ru Siberian Federal University; Institute of Biophysics SB RAS, Federal Research Center ‘Krasnoyarsk Science Center SB RAS’ ru Сибирский федеральный университет ru Siberian Federal University ru Сибирский федеральный университет ru Siberian Federal University ru Сибирский федеральный университет ru Siberian Federal University ru Сибирский федеральный университет ru Siberian Federal University ru Институт белка РАН ru Institute of Protein Research, Russian Academy of Sciences ru 25 09 2018 25 09 2018 3 3 484 488 20 09 2018 20 09 2018 https://rusjbpc.ru/en/nauka/article/54315/view

Исследование посвящено поиску новых информативных параметров для экспериментального определения структурных перестроек белков. Для этого было проведено сравнение кривых перехода, получаемых по характеристикам собственной флуоресценции белков при стационарных (непрерывное возбуждение) и время-разрешенных условиях (импульсное наносекундное возбуждение). Исследована равновесная денатурация мочевиной двух белков - карбоксиангидразы Б быка и бактериальной люциферазы, содержащих по семь триптофановых остатков. Проанализирована зависимость от концентрации мочевины следующих параметров флуоресценции: сдвиг спектра испускания при стационарных условиях, времена жизни, вклад спектральных компонент, ассоциированных с определённым временем жизни. Получено, что в случае карбоксиангидразы характеристики стационарной флуоресценции не отражают стадийность процесса денатурации, в отличие от параметров время-разрешенной флуоресценции. Это может быть связано с тем, что промежуточные состояния данного белка образуются при близких значениях концентрации мочевины. Стадии денатурации бактериальной люциферазы значительно разнесены по концентрации денатурирующего агента, поэтому они находят отражение, как в стационарных спектрах флуоресценции, так и в изменениях времен жизни. Обсуждается связь наблюдаемых различий в параметрах флуоресценции белков с локализацией их триптофановых остатков и общим механизмом денатурации.

The study contributes to the development of experimental methods capable of detecting the intermediate states of proteins upon equilibrium denaturation. The urea-induced transition curves of bovine carbonic anhydrase II and bacterial luciferase obtained from steady-state and time-resolved fluorescence were compared. The dependence of the following fluorescence parameters on the urea concentration was analyzed: the shift of the steady-state emission spectrum, the lifetimes, and the spectral contribution of the lifetime components. It was found that for carbonic anhydrase the steady-state fluorescence does not reflect the multi-stage denaturation, in contrast to the time-resolved fluorescence parameters. This can be due to the fact that the intermediate states of carbonic anhydrase are formed at close urea concentrations. For bacterial luciferase the transition midpoints of unfolding stages are significantly separated by urea concentration scale, so they can be seen both in steady-state fluorescence spectra and in lifetimes change. The observed differences in the fluorescence parameters of two proteins were discussed in terms of tryptophan residues location and the general mechanisms of unfolding pathways.

 собственная флуоресценция белков равновесная денатурация карбоксиангидраза Б бактериальная люцифераза время жизни флуоресценции путь денатурации белка intrinsic fluorescence of proteins equilibrium denaturation carbonic anhydrase II bacterial luciferase fluorescence lifetime protein unfolding pathway

Lakowicz J.R. Principles of fluorescence spectroscopy. New York: Springer Science, 2006. Lakowicz J.R. Principles of fluorescence spectroscopy. New York: Springer Science, 2006. Chen Y., Barkley M.D. Toward understanding tryptophan fluorescence in proteins. Biochemistry, 1998, vol. 37, pp. 9976-9982. Chen Y., Barkley M.D. Toward understanding tryptophan fluorescence in proteins. Biochemistry, 1998, vol. 37, pp. 9976-9982. Albani J.R. Origin of tryptophan fluorescence lifetimes. Part 1. Fluorescence lifetimes origin of tryptophan free in solution. J. Fluoresc., 2014, vol. 24, pp. 93-104. Albani J.R. Origin of tryptophan fluorescence lifetimes. Part 1. Fluorescence lifetimes origin of tryptophan free in solution. J. Fluoresc., 2014, vol. 24, pp. 93-104. Albani J.R. Origin of tryptophan fluorescence lifetimes. Part 2: Fluorescence lifetimes origin of tryptophan in proteins. J. Fluoresc., 2014, vol. 24, pp. 105-117. Albani J.R. Origin of tryptophan fluorescence lifetimes. Part 2: Fluorescence lifetimes origin of tryptophan in proteins. J. Fluoresc., 2014, vol. 24, pp. 105-117. Engelborghs Y. The analysis of time resolved protein fluorescence in multi-tryptophan proteins. Spectrochim. Acta A., 2001, vol. 57, pp. 2255-2270. Engelborghs Y. The analysis of time resolved protein fluorescence in multi-tryptophan proteins. Spectrochim. Acta A., 2001, vol. 57, pp. 2255-2270. Semisotnov G.V., Uversky V.N., Sokolovsky I.V., Gutin A.M., Razgulyaev O.I., Rodionova N.A. Two slow stages in refolding of bovine carbonic anhydrase B are due to proline isomerization. J. Mol. Biol., 1990, vol. 213, pp. 561-568. Semisotnov G.V., Uversky V.N., Sokolovsky I.V., Gutin A.M., Razgulyaev O.I., Rodionova N.A. Two slow stages in refolding of bovine carbonic anhydrase B are due to proline isomerization. J. Mol. Biol., 1990, vol. 213, pp. 561-568. Melnik B.S., Marchenkov V.V., Evdokimov S.R., Samatova E.N., Kotova N.V. Multy-state protein: Determination of carbonic anhydrase free-energy landscape. Biochem. Biophys. Res. Commun., 2008, vol. 369, pp. 701-706. Melnik B.S., Marchenkov V.V., Evdokimov S.R., Samatova E.N., Kotova N.V. Multy-state protein: Determination of carbonic anhydrase free-energy landscape. Biochem. Biophys. Res. Commun., 2008, vol. 369, pp. 701-706. Semisotnov G.V., Rodionova N.A., Kutyshenko V.P., Ebert B., Blanck J., Ptitsyn O.B. Sequential mechanism of refolding of carbonic anhydrase B. FEBS Lett., 1987, vol. 224, pp. 9-13. Semisotnov G.V., Rodionova N.A., Kutyshenko V.P., Ebert B., Blanck J., Ptitsyn O.B. Sequential mechanism of refolding of carbonic anhydrase B. FEBS Lett., 1987, vol. 224, pp. 9-13. Inlow J.K., Baldwin T.O. Mutational analysis of the subunit interface of Vibrio harveyi bacterial luciferase. Biochemistry, 2002, vol. 41, pp. 3906-3915. Inlow J.K., Baldwin T.O. Mutational analysis of the subunit interface of Vibrio harveyi bacterial luciferase. Biochemistry, 2002, vol. 41, pp. 3906-3915. Deeva A.A., Temlyakova E.A., Sorokin A.A., Nemtseva E.V., Kratasyuk V.A. Structural distinctions of fast and slow bacterial luciferases revealed by phylogenetic analysis. Bioinformatics, 2016, vol. 32, pp. 3053-3057. Deeva A.A., Temlyakova E.A., Sorokin A.A., Nemtseva E.V., Kratasyuk V.A. Structural distinctions of fast and slow bacterial luciferases revealed by phylogenetic analysis. Bioinformatics, 2016, vol. 32, pp. 3053-3057. Ameloot M., Hendrickx H. Extension of the performance of Laplace deconvolution in the analysis of fluorescence decay curves. Biophys. J., 1983, vol. 44, pp. 27-38. Ameloot M., Hendrickx H. Extension of the performance of Laplace deconvolution in the analysis of fluorescence decay curves. Biophys. J., 1983, vol. 44, pp. 27-38. Beechem J.M., Gratton E., Ameloot M., Knutson J.R., Brand L. The global analysis of fluorescence intensity and anisotropy decay data: second-generation theory and programs In Topics in fluorescence spectroscopy, Springer, Boston, MA, 2002, pp. 241-305. Beechem J.M., Gratton E., Ameloot M., Knutson J.R., Brand L. The global analysis of fluorescence intensity and anisotropy decay data: second-generation theory and programs In Topics in fluorescence spectroscopy, Springer, Boston, MA, 2002, pp. 241-305. Nemtseva E.V., Lashchuk O.O., Gerasimova M.A. Similarity of decay-associated spectra for tryptophan fluorescence of proteins with different structures. Biophysics, 2016, vol. 61, pp. 193-199. Nemtseva E.V., Lashchuk O.O., Gerasimova M.A. Similarity of decay-associated spectra for tryptophan fluorescence of proteins with different structures. Biophysics, 2016, vol. 61, pp. 193-199. Nemtseva E.V., Lashchuk O.O., Gerasimova M.A., Melnik T.N., Nagibina G.S., Melnik B.S. Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II. Methods and applications in fluorescence, 2018, vol. 6, pp. 015006. Nemtseva E.V., Lashchuk O.O., Gerasimova M.A., Melnik T.N., Nagibina G.S., Melnik B.S. Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II. Methods and applications in fluorescence, 2018, vol. 6, pp. 015006. Reshetnyak Y.K., Koshevnik Y., Burstein E.A. Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues. Biophys. J., 2001, vol. 81, pp. 1735-1758. Reshetnyak Y.K., Koshevnik Y., Burstein E.A. Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues. Biophys. J., 2001, vol. 81, pp. 1735-1758. Shen C., Menon R., Das D. [et al.] The protein fluorescence and structural toolkit: Database and programs for the analysis of protein fluorescence and structural data. Proteins, 2008, vol. 71, pp. 1744-1754. Shen C., Menon R., Das D. [et al.] The protein fluorescence and structural toolkit: Database and programs for the analysis of protein fluorescence and structural data. Proteins, 2008, vol. 71, pp. 1744-1754. Deeva A.A., Nemtseva E.V., Kratasyuk V.A. Structural properties of tryptophan microenvironment in bacterial luciferase. Luminesc., 2014, vol. 29, pp. 72-73. Deeva A.A., Nemtseva E.V., Kratasyuk V.A. Structural properties of tryptophan microenvironment in bacterial luciferase. Luminesc., 2014, vol. 29, pp. 72-73. Slyusareva E.A., Gerasimovа M.A., Sizykh A.G., Gornostaev L.M. Spectral and fluorescent indication of the acid-base properties of biopolymer solutions. Russ. Phys. J., 2011, vol. 54, pp. 485-492. Slyusareva E.A., Gerasimova M.A., Sizykh A.G., Gornostaev L.M. Spectral and fluorescent indication of the acid-base properties of biopolymer solutions. Russ. Phys. J., 2011, vol. 54, pp. 485-492. Tsuboi T., Penzkofer A., Slyusareva E., Sizykh A. Photoluminescence properties of fluorone dyes in bio-related films at low temperatures. J. Photochem. Photobiol. A., 2011, vol. 222, pp. 336-342. Tsuboi T., Penzkofer A., Slyusareva E., Sizykh A. Photoluminescence properties of fluorone dyes in bio-related films at low temperatures. J. Photochem. Photobiol. A., 2011, vol. 222, pp. 336-342.