Refolding of triosephosphate isomerase in low-water media investigated by fluorescence resonance energy transfer

Biochemistry
M A Sepúlveda-BecerraA Darszon

Abstract

The refolding and reassociation of rabbit muscle triosephosphate isomerase (TPI) monomers unfolded with guanidine hydrochloride (GdnHCl) was studied in aqueous media and in reverse micelles (RM) formed with hexadecyltrimethylammonium bromide and n-octane/hexanol. Fluorescence resonance energy transfer (FRET) studies were carried out using TPI labeled at Cys-217 with 5-((2-((iodoacetyl)-amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) and TPI labeled at one of the free amino groups with fluorescein 5'-isothiocyanate (FITC). Efficient FRET between monomers of AEDANS-TPI and FITC-TPI in aqueous media was detected 2-3 min after denaturant dilution and remained constant for hours. The distance between AEDANS and FITC in a labeled, renatured hetero-TPI dimer calculated from FRET results was 48 A, in reasonable agreement with estimates based on the crystal structure of TPI. In RM, recovery of enzyme activity during renaturation correlates with the appearance of a high-intrinsic fluorescence intermediate believed to be a partially folded monomer (Fernández-Velasco et al., 1995). Nevertheless, when AEDANS- and FITC-labeled monomers were mixed in RM, FRET occurred as soon as GdnHCl was diluted (FRET efficiency = 0.36), precedi...Continue Reading

References

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Citations

Feb 20, 2004·Journal of Colloid and Interface Science·Swati De, Agnishwar Girigoswami
Apr 17, 1998·Critical Reviews in Biochemistry and Molecular Biology·M Tuena de Gómez-Puyou, A Gómez-Puyou
Jun 23, 2009·Journal of Photochemistry and Photobiology. B, Biology·Deboleena SarkarNitin Chattopadhyay
Dec 21, 2012·Luminescence : the Journal of Biological and Chemical Luminescence·Pingping LiYouqiu He
Jan 27, 2015·Journal of Photochemistry and Photobiology. B, Biology·Fanbo ChenJihua Liu
Nov 4, 2016·Luminescence : the Journal of Biological and Chemical Luminescence·M G KotreshS R Inamdar
Aug 22, 1997·The Journal of Biological Chemistry·W Garzon-RodriguezC G Glabe

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