Investigación

 

The Equilibrium Unfolding of Triosephosphate Isomerase from T. cruzi in Guanidinium Hydrochloride is a four State Process. Intrinsic Fluorescence Studies

 

Edgar Vázquez-Contreras,*¹ Brenda Guadalupe Sánchez-Rebollar,¹ and María Elena Chánez-Cárdenas²

 

¹ Instituto de Química. Departamento de Bioquímica. Universidad Nacional Autónoma de México. Circuito Exterior, Ciudad Universitaria México, D.F. 04510, México. Telephone: (5255) 56224565. Fax: (5255) 56162217. E-mail: vazquezc@servidor.unam.mx.

² Laboratorio de Patología Vascular Cerebral, Instituto Nacional de Neurología y Neurocirugía "Manuel Velasco Suárez". México, D.F.

 

Recibido el 29 de septiembre del 2004.
Aceptado el 6 de diciembre del 2004.

 

Abstract

Equilibrium and kinetic folding pathways of several homologous proteins have been studied. Early studies concluded that the folding routes of homologous proteins follow fundamentally similar pathways, and that the folding of a certain conformation is conserved throughout evolution. However, there are examples of homologous proteins that unfold by different routes. Regarding triosephosphate isomerase (TIM), unfolding studies with enzymes from different sources, have shown: 1) two-state behaviors and 2) more complex processes (including two equilibrium-unfolding intermediates and inespecific aggregation), in the transition from de native homodimer to the denatured monomers. In this work, we studied the changes in intrinsic fluorescence of TIM from Trypanosoma cruzi after incubation in guanidinium hydrochloride. Our results show that the reaction is described by a four state process. Finally we discuss the results in terms of the heterogeneity observed in TIM denaturation.

Key words: Triosephoshate isomerase, protein folding equilibrium intermediates.

 

Resumen

Actualmente existen diversos estudios cinéticos y al equilibrio de proteínas homólogas. Los primeros análisis al respecto concluyeron que las rutas de plegamiento siguen caminos similares para estas proteínas y que ciertas conformaciones se conservan durante la evolución. Sin embargo, existen proteínas homólogas que se pliegan por vías diferentes. Con respecto a los estudios realizados con la triosafosfato isomerasa (TIM) la desnaturalización ha mostrado: 1) procesos de dos estados y 2) procesos más complejos (incluyendo dos intermediarios y agregación inespecífica), en la transición del homodímero nativo a los monómeros desnaturalizados. En este trabajo estudiamos los cambios de la fluorescencia intrínseca de la TIM de Trypanosoma cruzi después de la incubación en hidrocloruro de guanidina. Nuestros resultados muestran que la reacción es descrita por un proceso de cuatro estados. Finalmente discutimos los resultados en términos de la heterogeneidad observada en la desnaturalización de la TIM.

Palabras clave: Triosafosfato isomerasa, plegamientos intermediarios al equilibrio.

 

Dedicated to the memory of Dr. Raymundo Cruz Almanza.

 

Introduction

Equilibrium and kinetic folding pathways of several homologous proteins have been characterized. Early studies concluded that the folding routes of homologous proteins follow fundamentally similar paths and that the folding of a certain conformation is conserved throughout evolution [1-4]. However, there are examples of homologous proteins which unfold with different patterns [5,6]. Regarding triosephosphate isomerase (TIM) unfolding and refolding from several species has been studied extensively [7-25]. Thermodynamic data for unfolding have been reported only for human, rabbit and yeast enzymes [8,17-19], where a three-state model (N ⇆ 2M ⇆ 2U) has been proposed. In human TIM (hTIM), the unfolding of the wild-type enzyme was modeled as a two-state transition, and information on the properties of the monomer was obtained from the two-state unfolding of monomeric mutants [8,18]. The stability of the TIM homodimer was calculated for rabbit TIM from equilibrium and activation energies for the wild-type enzyme [17]. The same unfolding behavior was found with yeast TIM (yTIM) [9,10,19], although in yTIM was first evidenced the presence of an stable equilibrium intermediate in the unfolding of the triosephosphate isomerase [9]. For yTIM the thermodynamic of the process was obtained for the denaturation and renaturation reactions [19]. In the present work we show that following changes in intrinsic fluorescence parameters of Trypanosoma cruzi TIM (TcTIM) induced by guanidinium hydrochoride (Gdn-HCl), the transition between the homodimeric enzyme and the unfolded monomers follows a four-state process.

 

Results and discussion

Changes in tertiary structure of TcTIM after equilibrium unfolding

To characterize the equilibrium unfolding of the homodimer of Triosephosphate isomerase from Trypanosoma cruzi (TcTIM) changes in intrinsic fluorescence spectra was studied at increasing guanidinium hydrochloride (Gdn-HCl) concentrations. In an average protein¹ 8.5 % of the total amino acids residues are aromatic [26]. In TcTIM every monomer is composed by 251 amino acids residues, 18 of which are aromatics (7.17 %), 5 are tryptophan (1.99 %), 6 tyrosine (2.39 %) and 7 phenylalanine (2.79 %) residues (Figure 1). This number of fluorescent residues is enough to adequately study fluorescence changes in this enzyme as a function of denaturant concentration.

We studied the change in intrinsic fluorescence parameters i.e. spectral center of mass (SCM), wavelength of maximal emission (λ_máx), maximum fluorescence intensity (FI) and fluorescence intensity at native λ_máx (FI-λ_máx) (see below), after exposition of W, Y and F residues (exciting the samples at 280 nm) and after the exposition of only W residues (exciting the samples at 295 nm) that occurs during the dissociation and/or unfolding of the homodimer of TcTIM, we use this evidence as a prove of protein packing.

The equilibrium denaturation of TcTIM (50 µg mL^-1) was induced by the addition of different concentrations of guanidinium hydrochloride (Gdn-HCl) at 25 °C for equilibrium time (48 h) [20]. After incubation of the homodimer Gdn-HCl induces a red shift in SCM and λ_máx (Table 1). As the Gdn-HCl concentration increases, the unfolding of TcTIM produced a nonmonophasic pattern; this behavior is observed both when following the exposure of WYF residues and when following only the exposure of W residues. This behavior is observed for all the spectroscopic characteristics obtained from fluorescence spectra i.e. SCM, λ_máx, FI and FI-λ_máx (Table 1). This result indicates that the denaturation process of TcTIM is a non two-state process as suggested previously [20].

Four state model of denaturation of TcTIM

The analysis of the results presented here suggest that the model that describes de denaturation pattern of TcTIM is a four state process. This reaction departs with the native enzyme (N₂) which at low Gdn-HCl concentrations (≈ 0.5 M) partially unfold to produce a first intermediate (I₁). After excitation at both 295 or 280 nm, this conformer show an increase in FI relative to native enzyme (Table 1). This increase in FI has not been no reported for any other TIM denaturation, and it may be explained by either the effect of ionic strength on the fluorescence intensity or by the change in polar surroundings of the aromatic residues of the protein at low Gdn-HCl concentration. At medium denaturant concentrations (1.0 - 2.0 M Gdn-HCl) the first intermediate partially unfolds to produce a second equilibrium conformer (I₂) characterized by a plateau region in the unfolding pattern. At high denaturant concentrations I₂ totally unfolds to produce the unfolded monomers (2U). From the overall observation in changes in intrinsic fluorescence, the suggested pattern for TcTIM equilibrium denaturation in Gdn-HCl is described by:

The equilibrium denaturation of homologous TIMs

The amino acid sequences and the three dimensional structures of oligomeric TIM from different species are similar. In spite of these structural similarities, the equilibrium-unfolding studies with enzymes from different species show heterogeneous behaviour. As we report here for the equilibrium unfolding of TcTIM, the four state behavior was earlier described for the denaturation with Gdn-HCl of TIM from Plasmodium falciparum [11]. In this process one of the states is described as an aggregate and the process is irreversible. This fact complicates the study of the thermodynamics of the unfolding. The equilibrium unfolding of TIM from T. brucei (TbTIM) is even more complex [21], in this transition the irreversible aggregation of both non-native dimer and monomeric expanded intermediates was detected. Three-state process with a monomeric intermediate was reported for the enzyme of yTIM [9,10,19]. Two-state process has been observed for the enzymes of B. stearothermophilus (BsTIM) [18], rabbit (rTIM) [17,22,23] and L. mexicana (LmTIM) [12].

In the extensively studied unfolding of TIM, irreversibility has been often observed [11,21,24,25]; on the other hand, for TcTIM the renaturation reaction is fully reversible [28]. This observation suggests that in TcTIM unfolding induced by Gdn-HCl, the observed intermediates are not aggregates. This conclusion is important because the denaturation pattern of the triosephosphate isomerase from T. brucei [21] (TbTIM) a notably similar protein to TcTIM [20] is irreversible. In TbTIM denaturation induced by Gdn-HCl the intermediates where characterized as a non native dimer and as expanded monomer, it was also demonstrated that both intermediates are prone to aggregate. We are performing experiments in order to characterize the nature of I₁ and I₂. This information allows us to construct a model to address the thermodynamic characterization of the process.

Final remarks

Even the final folding and activity are conserved in TIM enzyme, the changes in the amino acids sequence causes very heterogeneous unfolding patterns in enzymes from different species. Even so, and despite the irreversibility and complexity often found in the unfolding of oligomeric proteins, the study of the energetic properties of these biomolecules are necessary because of the constant participation of oligomers in metabolism.

 

Experimental

Overexpression and purification of recombinant protein

E.coli BL21DE3 cells with recombinant TcTIM gene were a generous gift of Dr. Ruy Pérez-Montfort (Instituto de Fisiología Celular, UNAM, México). Overexpression and purification of TcTIM was performed as described by Ostoa-Saloma [29]. Purified fractions showed a single band by SDS-PAGE.

Gdn-HCl unfolding experiments monitored by fluorescence

Denaturation experiments were performed incubating 50 µg mL^-1 of TcTIM at 25 °C in 100 mM TEA /10 mM EDTA /1.0 mM DTT, pH 7.4 for at least 48 h. Gdn-HCl concentration was increased from 0 to 6.0 M. The fluorescence changes were performed using an ISS PCI Photon Counting Spectrofluorometer (ISS, Urbana, IL) at 25 °C using both excitation wavelengths 280 and 295 nm (slit width = 1 mm). The spectral center of mass (SCM) of each spectrum defined as

was calculated with the ISS software. In equation (2) I(λ) is the fluorescence intensity at wavelength λ. Reference samples were subtracted from all the experimental measurements.

 

Acknowledgments

This work was supported by Grants 40524M and 41328Q from CONACyT. B.G.S-R is the recipient of fellowship from CONACyT. We thank Laboratorio de Fisicoquímica y Diseño de Proteínas, Facultad de Medicina, and Dr A. Gómez-Puyou and Marietta Tuena, IFC UNAM for generously making available their equipment facilities. We are grateful to Beatriz Aguirre and Dr. Gerardo Pérez-Hernández for his help in TcTIM purification.

 

References

1. Hollecker. M.; Creighton, T. E. J. Mol. Biol. 1983, 168, 409-437.         [ Links ]

2. Krebs H, Schmid FX, Jaenicke R. J. Mol. Biol. 1983, 169, 619-635.         [ Links ]

3. Stackhouse TM, Onuffer JJ, Matthews R, Ahmed SA, Miles EW. Biochemistry 1988, 27, 824-832.         [ Links ]

4. Kragelund, BB; Hojrup, P; Jensen, M. S.; Schjerling, C. K.; Juul, E.; Knudsen. J.; Poulsen, F.M. J. Mol. Biol. 1996, 256, 187-200.         [ Links ]

5. Ferguson, N.; Capaldi, A.P.; James, R.; Kleanthous, C.; Radford, S.E.; J. Mol. Biol. 1999, 286,1597-1608.         [ Links ]

6. Zerovnik, E.; Virden, R.; Jerala, R.; Turk, V.; Waltho, J.P. Proteins 1998, 32, 296-303.         [ Links ]

7. Zabori, S.; Rudolph, R.; Jaenicke, R. Z. Naturforsch. 1980, 35c, 999-1004.         [ Links ]

8. Mainfroid, V.; Terpstra, P.; Beauregard, M.; Frère, J.M.; Mande, S.C.; Hol, W.G.J.; Martial, J.A.; Goraj, K. J. Mol. Biol. 1996, 257, 441-456.         [ Links ]

9. Vázquez-Contreras, E.; Zubillaga, R.; Mendoza-Hernández, G.; Costas, M.; Fernández-Velasco, D. A. Protein Peptide Lett. 2000, 7, 57-64.         [ Links ]

10. Morgan, C. J.; Wilkins, D. K.; Smith, L. J.; Kawata, Y.; Dobson, C. M. J. Mol. Biol. 2000 300, 11-16.         [ Links ]

11. Gokhale, R. S.; Ray, S. S.; Balaram, H.; and Balaram, P. Biochemistry 1999 38, 423-431.         [ Links ]

12. Lambeir, A. M.; Backmann, J.; Ruiz-Sanz, J.; Filimonov, V.; Nielsen, J. E.; Kursula, I.; Norledge, B. V.; Wierenga, R. K. Eur. J. Biochem. 2000 267, 2516-2524.         [ Links ]

13. Borchert, T.V.; Abagyan, R.; Jaenicke, R.; Wierenga, R.K. Proc. Natl. Acad. Sci. USA, 1994, 91, 1515-1518.         [ Links ]

14. Schliebs, W.; Thanki, N.; Jaenicke, R.; Wierenga, R.K. Biochemistry 1997, 36, 9655-9662.         [ Links ]

15. Fernández-Velasco, D.A.; Sepúlveda-Becerra, M.; Galina, A.; Darszon, A.; Tuena de Gómez-Puyou, M.; Gómez-Puyou, A. Biochemistry 1995, 34, 361-369.         [ Links ]

16. Waley, S. Biochem. J. 1973, 135, 165-172.         [ Links ]

17. Rietveld, A. W.; Ferreira, S. T. Biochemistry 1998, 35, 7743-7751.         [ Links ]

18. Mainfroid, V.; Mande, S.C.; Hol, W.G.J.; Martial, J.A.; Goraj, K. Biochemistry 1996, 35, 4110-4117.         [ Links ]

19. Najera, M.; Costas, M.; Fernández-Velasco, D. A. Biochem. J. 2003, 370, 785-792.         [ Links ]

20. Chánez-Cárdenas, M. E.; Vázquez-Contreras, E. Rev. Soc. Quím. Méx. 2002, 46, 3, 219-222.         [ Links ]

21. Chánez-Cárdenas, M. E.; Fernández-Velasco, D. A.; Vázquez-Contreras, E.; Coria, R.; Saab-Rincón, G.; Pérez-Montfort, R. Arch. Biochem. Biophys. 2002, 399, 117-129.         [ Links ]

22. Moreau, V. H.; Rietveld, A.W. M.; Ferreira S. T. Biochemistry 2003, 42, 14831-14837.         [ Links ]

23. Pan, H.; Raza, A. S.; Smith, D. L. J. Mol. Biol. 2004, 336, 1251-1263.         [ Links ]

24. Alvarez, M.; Zeelen, J. P.; Mainfroid, V.; Rentier-Delrue, F.; Martial, J. A.; Wyns, L.; Wierenga, R. K.; Maes, D. J. Biol. Chem. 1998, 273, 2199-2206.         [ Links ]

25. Beaucamp, N.; Hofmann, A.; Kellerer, B.; Jaenicke, R. Protein Sci. 1997, 6, 2159-2165.         [ Links ]

26. Doolittle, R.F. Redundante in protein sequences. In prediction of protein structure and the principles of protein conformation (Fastman, G.D eds.) Plenum Press NY, 1989, pp 599-623.         [ Links ]

27. Maldonado, E.; Soriano-García, M.; Moreno, A.; Cabrera, N.; Garza-Ramos, G.; Tuena de Gómez-Puyou, M.; Gómez-Puyou, A.; Pérez-Montfort, R. J. Mol. Biol. 1998, 283, 193-203.         [ Links ]

28. Zomosa-Signoret, V.; Hernández-Alcántara, G.; Reyes-Vivas, H.; Martínez-Martínez, E.; Garza-Ramos, G.; Pérez-Montfort, R.; Tuena de Gómez-Puyou, M.; Gómez-Puyou, A. Biochemistry 2003, 42, 331.         [ Links ]

29. Ostoa-Saloma, P.; Garza-Ramos, G.; Ramírez, J.; Becker, I.; Berzunza, I.; Landa, A.; Gómez-Puyou, A.; Tuena de Gómez-Puyou, M.; Pérez-Montfort, R. Eur. J. Biochem. 1997, 244, 700-705.         [ Links ]

 

Nota

¹ Average occurrence in over 1500 different proteins.