proteins STRUCTURE O FUNCTION O BIOINFORMATICS The folding pathway of glycosomal triosephosphate isomerase: Structural insights into equilibrium intermediates Valeria Guzman-Luna* and Georgina Garza-Ramos Laboratorio de Fisicoquı ´mica e Ingenierı´a de Proteı´nas, Departamento de Bioquı´mica, Facultad de Medicina, Universidad Nacional Auto ´noma de Me ´xico, Ciudad Universitaria. Me ´xico, D.F. 04510, Me ´xico INTRODUCTION Studying equilibrium folding pathways can reveal the principles that dictate the acquisition of a biologically relevant protein conformation. The dimeric triosephosphate isomerase of Typanosoma brucei (TbTIM) is an ideal model for studying the energetic contribution of quaternary interactions to the conformational stability of a (b/a) 8 barrel enzyme, which is the most common fold found in nature. Triosephosphate isomerase (TIM) is a ubiquitous enzyme that catalyzes the fifth reac- tion in glycolysis. In mesophiles, the catalytically active conformation is a homodimer that depends on quaternary interactions to carry out the isomerization reaction. The typical (b/a) 8 -barrel contains approxi- mately 200–260 amino acids. In TbTIM, each subunit is composed of 250 residues. TbTIM must be located in the glycosome to carry out the physiological function, as the expression of TIM in the cytosol of T. brucei inhibits parasite growth. 1 Thus far, TIM is the only enzyme of T. brucei that is known to be devoid of the consensus motifs for either of the two reported peroxisome-targeting signals that are essential for glycosomal localization. Instead, TbTIM has an internal fragment of 22 amino acids (from 140 to 161) that transfers the protein inside glycosomes of T. brucei. 2 Approximately, 10% of all known protein structures contain at least one (b/a) 8 barrel domain, which is also known as the TIM bar- rel. Not surprisingly, experimental and computational work has focused on the viability of this scaffolding for enzyme design. 3 A TIM barrel consists of an eightfold repeat of a b-strand-loop-a-helix (ba) motif. The eight b-strands form a central parallel b-sheet, which constitutes the major hydrophobic core, whereas the amphi- pathic a-helices form the external envelop. In all known TIM barrels, the catalytic residues are located at the C-terminal ends of the b-strands and in the b/a-loops. Our current understanding of the molecular folding mechanisms of TIM barrels is largely based on Additional Supporting Information may be found in the online version of this article. Abbreviations: a-GDH, a-glycerol-3-phosphate dehydrogenase; GuHCl, guanidine hydrochloride; NADH, nicotinamide adenine dinucleotide hydride; PGA, 2-phosphoglycolate; TbTIM, Trypano- soma brucei TIM; TEA, triethanolamine; TIM, triosephosphate isomerase. Grant sponsor: CONACyT; Grant number: 41328. *Correspondence to: Valeria Guzman-Luna, Laboratorio de Fisicoquı´mica e Ingenierı´a de Proteı´nas, Departamento de Bioquı´mica, Facultad de Medicina, Universidad Nacional Auto ´noma de Me ´xico, Ciudad Universitaria. Me ´xico, D.F. 04510, Me ´xico. E-mail: valguz@laguna.fmedic.unam.mx Received 7 September 2011; Revised 9 February 2012; Accepted 23 February 2012 Published online 12 March 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ prot.24063 ABSTRACT The guanidine hydrochloride-induced conforma- tional 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 sta- bility parameters of TIM are as follows: DG I2-N2 5 5.5 6 0.6, DG 2M-I2 519.6 6 1.6, and DG 2U-2M 5 14.7 6 3.1 kcal mol 21 . The I 2 state is compact (a SR 5 0.8); it is able to bind 8-anilinonaphtha- lene-1-sulfonic acid ANS and it is composed of 45% of a-helix and tertiary structure content compared with the native enzyme; however, it is unable to bind the transition-state analog 2-phos- phoglycolate. Conversely, the 2M state lacks de- tectable tertiary contacts, possesses 10% of the native a-helical content, is significantly expanded (a SR 5 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 b 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, indi- cating solvent exposure of W90 at b 4 . Our results suggest that the I 2 state comprises the (ba) 1-4 b 5 module in which the conserved C126 residue located at b 5 defines the boundary of the folded segment. We propose a folding pathway that high- lights the remarkable thermodynamic stability of this glycosomal enzyme. Proteins 2012; 00:000–000. V V C 2012 Wiley Periodicals, Inc. Key words: hydrophobic clusters; kinetic stabil- ity; partially folded conformations; thermody- namic stability; TIM barrels. V V C 2012 WILEY PERIODICALS, INC. PROTEINS 1