title: Refolding of triosephosphate isomerase in low-water media investigated by fluorescence resonance energy transfer.
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), preceding the changes in TPI intrinsic fluorescence and reactivation. Thereafter, the efficiency of FRET increased during the next hour up to approximately 0.50, where it remained after 24 h, when 80% of the enzyme activity was recovered. The high initial FRET seen in RM could indicate the formation of an inactive dimer within the first minutes after denaturant dilution. The further increase in FRET observed over the next hour could reflect conformational rearrangements of the protein and transition from the inactive to the active dimer.
authors.affiliation: Départamento Genética y Fisiología Molecular, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.
authors.forename: M A
authors.initials: MA
authors.lastname: Sepúlveda-Becerra
authors.forename: S T
authors.initials: ST
authors.lastname: Ferreira
authors.forename: R J
authors.initials: RJ
authors.lastname: Strasser
authors.forename: W
authors.initials: W
authors.lastname: Garzón-Rodríguez
authors.forename: C
authors.initials: C
authors.lastname: Beltrán
authors.forename: A
authors.initials: A
authors.lastname: Gómez-Puyou
authors.forename: A
authors.initials: A
authors.lastname: Darszon
mesh_terms: D000818:Animals; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D000441:Hexanols / Q000378:metabolism; D008823:Micelles; D018482:Muscle, Skeletal / Q000201:enzymology; D009282:Naphthalenesulfonates / Q000378:metabolism; D009795:Octanes / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002593:Cetrimonium Compounds; D003902:Detergents; D006146:Guanidines; D000441:Hexanols; D008823:Micelles; D009282:Naphthalenesulfonates; D009795:Octanes; C006421:1,5-I-AEDANS; D014305:Triose-Phosphate Isomerase; D016650:Fluorescein-5-isothiocyanate; D019791:Guanidine; D003545:Cysteine; C026728:octane; D000077286:Cetrimonium