Persistent conformational heterogeneity of triosephosphate isomerase: separation and characterization of conformational isomers in solution.
Subunit dissociation of dimeric rabbit muscle triosephosphate isomerase (TIM) by hydrostatic pressure has previously been shown not to follow the expected dependence on protein concentration [Rietveld and Ferreira (1996) Biochemistry 35, 7743-7751]. This anomalous behavior was attributed to persistent conformational heterogeneity (i.e., the coexistence of long-lived conformational isomers) in the ensemble of TIM dimers. Here, we initially show that subunit dissociation/unfolding of TIM by guanidine hydrochloride (GdnHCl) also exhibits an anomalous dependence on protein concentration. Dissociation/unfolding of TIM by GdnHCl was investigated by intrinsic fluorescence and circular dichroism spectroscopies and was found to be a highly cooperative transition in which the tertiary and secondary structures of the protein were concomitantly lost. A procedure based on size-exclusion chromatography in the presence of intermediate (0.6 M) GdnHCl concentrations was developed to isolate two conformational isomers of TIM that exhibit significantly different stabilities and kinetics of unfolding by GdnHCl. Complete unfolding of the two isolated conformers at a high GdnHCl concentration (1.5 M), followed by refolding by removal of the denaturant, completely abolished the differences in their unfolding kinetics. These results indicate that such differences stem from conformational heterogeneity of TIM and are not related to any chemical modification of the protein. Furthermore, they add support to the notion that long-lived conformational isomers of TIM coexist in solution and provide a basis for the interpretation of the persistent heterogeneity of this protein.
Moreau
Vitor Hugo
VH
Departamento de Bioquímica Médica, ICB, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21994-590, Brazil.
Rietveld
Alex W M
AW
Ferreira
Sergio T
ST
D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019791:Guanidine / Q000737:chemistry; D006874:Hydrostatic Pressure; D007536:Isomerism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D011817:Rabbits; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000302:isolation & purification
D021122:Protein Subunits; D012996:Solutions; D014305:Triose-Phosphate Isomerase; D019791:Guanidine