Three-dimensional solution structure and stability of thioredoxin m from spinach
Proton NMR spectral resonances of thioredoxin m from spinach have been assigned, and its solution structure has been determined on the basis of 1156 nuclear Overhauser effect- (NOE-) derived distance constraints by using restrained molecular dynamics calculations. The average pairwise root-mean-squa...
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| Autores principales: | , , , , |
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| Formato: | JOUR |
| Materias: | |
| Acceso en línea: | http://hdl.handle.net/20.500.12110/paper_00062960_v40_n50_p15246_Neira |
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| Sumario: | Proton NMR spectral resonances of thioredoxin m from spinach have been assigned, and its solution structure has been determined on the basis of 1156 nuclear Overhauser effect- (NOE-) derived distance constraints by using restrained molecular dynamics calculations. The average pairwise root-mean-square deviation (RMSD) for the 25 best NMR structures for the backbone was 1.0 ± 1, when the structurally well-defined residues were considered. The N- and C-terminal segments (1-13 and 118-119) and residues 41-49, comprising the active site, are highly disordered. At the time of concluding this work, a crystal structure of this protein was reported, in which thioredoxin m was found to crystallize as noncovalent dimers. Although the solution and crystal structures are very similar, no evidence was found about the existence of dimers in solution, thus confirming that dimerization is not needed for the regulatory activity of thioredoxin m. The spinach thioredoxin m does not unfold by heat in the range 25-85 °C, as revealed by thermal circular dichroic (CD) measurements. However, its unfolding free energy (9.1 ± 0.8 kcal mol-1, at pH 5.3 and 25 °C) could be determined by extrapolating the free energy values obtained at different concentrations of guanidinium chloride (GdmC). The folding-unfolding process is two-state as indicated by the coincidence of the CD denaturation curves obtained at far and near UV. The H/D exchange behavior of backbone amide protons was analyzed. The slowest-exchanging protons, requiring a global-unfolding mechanism in order to exchange, are those from β2, β3, and β4, the central strands of the β-sheet, which constitute the main element of the core of the protein. The free energies obtained from exchange measurements of protons belonging to the α-helices are lower than those derived from GdmCdenaturation studies, indicating that those protons exchange by local-unfolding mechanisms. |
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