Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of alpha-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.

Stella, L., Caccuri, A.m., Rosato, N., Nicotra, M., LO BELLO, M., DE MATTEIS, F., et al. (1998). Flexibility of helix 2 in the human glutathione transferase P1-1. time-resolved fluorescence spectroscopy. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 273(36), 23267-23273 [10.1074/jbc.273.36.23267].

Flexibility of helix 2 in the human glutathione transferase P1-1. time-resolved fluorescence spectroscopy

STELLA, LORENZO;CACCURI, ANNA MARIA;ROSATO, NICOLA;LO BELLO, MARIO;DE MATTEIS, FABIO;FEDERICI, GIORGIO;RICCI, GIORGIO
1998-09-04

Abstract

Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of alpha-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.
4-set-1998
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore BIO/10 - BIOCHIMICA
English
Con Impact Factor ISI
mutagenesis, site-directed; glutathione s-transferase pi; protein structure, secondary; pliability; spectrometry, fluorescence; models, molecular; glutathione; humans; isoenzymes; glutathione transferase; models, chemical; binding sites
Stella, L., Caccuri, A.m., Rosato, N., Nicotra, M., LO BELLO, M., DE MATTEIS, F., et al. (1998). Flexibility of helix 2 in the human glutathione transferase P1-1. time-resolved fluorescence spectroscopy. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 273(36), 23267-23273 [10.1074/jbc.273.36.23267].
Stella, L; Caccuri, Am; Rosato, N; Nicotra, M; LO BELLO, M; DE MATTEIS, F; Mazzetti, A; Federici, G; Ricci, G
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/69136
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