Sensitivity of superfolder GFP to ionic agents

Olesya V. Stepanenko, Olga V. Stepanenko, Irina M. Kuznetsova, Vladislav V. Verkhusha, Konstantin K. Turoverov

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

Superfolder variant of the green fluorescent protein (sfGFP) became a favorite probe for examination of the unfolding-refolding processes of fluorescent proteins with beta-barrel structure owing to its reversible unfolding in comparison with other fluorescent proteins. Its benefit is the proper folding even in fusion constructions with poorly folded polypeptides. We noticed that guanidine thiocyanate affects not only the structure of protein but its chromophore directly. Therefore we studied the influence of ionic denaturants and salts including guanidine thiocyanate, guanidine hydrochloride, sodium chloride and sodium thiocyanate on spectral features of sfGFP. It was shown that moderate amounts of the studied agents do not disrupt sfGFP structure but provoke pronounced alteration of its spectral characteristics. Changes in absorption and CD spectra in visible spectral range indicate the specific binding of SCN- and Cl- anions in the sfGFP chromophore vicinity. The anion binding results in the redistribution of sfGFP molecules with neutral and anionic chromophores. This also hinders the proton transfer in the chromophore excited state, considerably decreasing the fluorescence intensity of sfGFP. Our results indicate that when ionic denaturants are used in the studies of fluorescent protein folding their effect on fluorophore charge state should be taken into account.

Original languageEnglish (US)
Article numbere110750
JournalPloS one
Volume9
Issue number10
DOIs
StatePublished - Oct 27 2014

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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    Stepanenko, O. V., Stepanenko, O. V., Kuznetsova, I. M., Verkhusha, V. V., & Turoverov, K. K. (2014). Sensitivity of superfolder GFP to ionic agents. PloS one, 9(10), [e110750]. https://doi.org/10.1371/journal.pone.0110750