The folding pathway of glycosomal triosephosphate isomerase: structural insights into equilibrium intermediates.

ABSTRACT

The guanidine hydrochloride-induced conformational transitions of glycosomal triosephosphate isomerase (TIM) were monitored with functional, spectroscopic, and hydrodynamic measurements. The equilibrium folding pathway was found to include two intermediates (N(2) ↔I(2) ↔2M↔2U). According to this model, the conformational stability parameters of TIM are as follows ΔG(I2-N2) = 5.5 ± 0.6, ΔG(2M-I2) =19.6 ± 1.6, and ΔG(2U-2M) = 14.7 ± 3.1 kcal mol(-1) . The I(2) state is compact (α(SR) = 0.8); it is able to bind 8-anilinonaphthalene-1-sulfonic acid ANS and it is composed of ∼45% of α-helix and tertiary structure content compared with the native enzyme; however, it is unable to bind the transition-state analog 2-phosphoglycolate. Conversely, the 2M state lacks detectable tertiary contacts, possesses ∼10% of the native α-helical content, is significantly expanded (α(SR) = 0.2), and has low affinity for ANS. We studied the effect of mutating cysteine residues on the structure and stability of I(2) and 2M. Three mutants were made C39A, C126A, and C39A/C126A. The replacement of C39, which is located at ß(2) , was found to be neutral. The I(2) -C126A state, however, was prone to aggregation and exhibited an emission maximum that was 3-nm red-shifted compared with the I(2) -wild type, indicating solvent exposure of W90 at ß(4) . Our results suggest that the I(2) state comprises the (ßα)(1-4) ß(5) module in which the conserved C126 residue located at ß(5) defines the boundary of the folded segment. We propose a folding pathway that highlights the remarkable thermodynamic stability of this glycosomal enzyme.