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The folding pathway of glycosomal triosephosphate isomerase: Structural insights into equilibrium intermediates
Author(s) -
GuzmanLuna Valeria,
GarzaRamos Georgina
Publication year - 2012
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.24063
Subject(s) - triosephosphate isomerase , chemistry , guanidine , folding (dsp implementation) , protein folding , isomerase , protein tertiary structure , stereochemistry , cysteine , crystallography , protein structure , enzyme , biochemistry , electrical engineering , engineering
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: Δ G I2‐N2 = 5.5 ± 0.6, Δ G 2M‐I2 =19.6 ± 1.6, and Δ G 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 β 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 β 4 . Our results suggest that the I 2 state comprises the (βα) 1‐4 β 5 module in which the conserved C126 residue located at β 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; © 2012 Wiley Periodicals, Inc.