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

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 (N2?I2?2M?2U). According to this model, the conformational stability parameters of TIM are as follows: ?GI2-N2 = 5.5 +/- 0.6, ?G2M-I2=19.6 +/- 1.6, and ?G2U-2M = 14.7 +/- 3.1 kcal mol-1. The I2 state is compact (aSR = 0.8); it is able to bind 8-anilinonaphthalene-1-sulfonic acid ANS and it is composed of similar to 45% of a-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 similar to 10% of the native a-helical content, is significantly expanded (aSR = 0.2), and has low affinity for ANS. We studied the effect of mutating cysteine residues on the structure and stability of I2 and 2M. Three mutants were made: C39A, C126A, and C39A/C126A. The replacement of C39, which is located at beta 2, was found to be neutral. The I2C126A state, however, was prone to aggregation and exhibited an emission maximum that was 3-nm red-shifted compared with the I2wild type, indicating solvent exposure of W90 at beta 4. Our results suggest that the I2 state comprises the (beta a)1-4 beta 5 module in which the conserved C126 residue located at beta 5 defines the boundary of the folded segment. We propose a folding pathway that highlights the remarkable thermodynamic stability of this glycosomal enzyme. Proteins 2012; (c) 2012 Wiley Periodicals, Inc.