Unfolding of triosephosphate isomerase from Trypanosoma brucei: identification of intermediates and insight into the denaturation pathway using tryptophan mutants.
The unfolding of triosephosphate isomerase (TIM) from Trypanosoma brucei (TbTIM) induced by guanidine hydrochloride (GdnHCl) was characterized. In contrast to other TIMs, where unfolding is a two or three state process, TbTIM showed two intermediates. The solvent exposure of different regions of the protein in the unfolding process was characterized spectroscopically with mutant proteins in which tryptophans (W) were changed to phenlylalanines (F). The midpoints of the transitions measured by circular dichroism, intrinsic fluorescence, and catalytic activity, as well as the increase in 1-aniline 8-naphthalene sulfonate fluorescence, show that the native state was destabilized in the W12F and W12F/W193F mutants, relative to the wild-type enzyme. Using the hydrodynamic profile for the unfolding of a monomeric TbTIM mutant (RMM0-1TIM) measured by size-exclusion chromatography as a standard, we determined the association state of these intermediates: D*, a partially expanded dimer, and M*, a partially expanded monomeric intermediate. High-molecular-weight aggregates were also detected. At concentrations over 2.0 M GdnHCl, the hydrodynamic properties of TbTIM and RMM0-1TIM are the same, suggesting that the dimeric intermediate dissociates and the unfolding proceeds through the denaturation of an expanded monomeric intermediate. The analysis of the denaturation process of the TbTIM mutants suggests a sequence for the gradual exposure of W residues: initially the expansion of the native dimer to form D* affects the environments of W12 and W159. The dissociation of D* to M* and further unfolding of M* to U induces the exposure of W170. The role of protein concentration in the formation of intermediates and aggregates is discussed considering the irreversibility of this unfolding process.
Chánez-Cárdenas
María Elena
ME
Departamento de Bioquímica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F., México.
Fernández-Velasco
D Alejandro
DA
Vázquez-Contreras
Edgar
E
Coria
Roberto
R
Saab-Rincón
Gloria
G
Pérez-Montfort
Ruy
R
D000818:Animals; D002384:Catalysis; D002942:Circular Dichroism; D005453:Fluorescence; D019791:Guanidine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D014364:Tryptophan; D014305:Triose-Phosphate Isomerase; D019791:Guanidine