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Abstract:

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.

Registro:

Documento: Artículo
Título:Three-dimensional solution structure and stability of thioredoxin m from spinach
Autor:Neira, J.L.; González, C.; Toiron, C.; De Prat-Gay, G.; Rico, M.
Filiación:Centro de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain
Instituto de Estructura de la Materia, CSIC, 28006 Madrid, Spain
Instituto de Investigaciones Bioquimicas, Fundacion Campomar and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Patricias Argentinas 435, 1405 Buenos Aires, Argentina
Palabras clave:Three-dimensional solutions; Crystal structure; Crystallization; Dimers; Extrapolation; Free energy; Ion exchange; Molecular dynamics; Nuclear magnetic resonance spectroscopy; pH effects; Protons; Proteins; thioredoxin; thioredoxin m; unclassified drug; article; circular dichroism; crystal structure; molecular dynamics; nonhuman; nuclear magnetic resonance; priority journal; protein conformation; protein denaturation; protein stability; protein tertiary structure; spinach; structure analysis; Amides; Amino Acid Sequence; Circular Dichroism; Crystallization; Crystallography, X-Ray; Dimerization; Drug Stability; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Protein Folding; Protein Structure, Secondary; Protons; Solutions; Spinacia oleracea; Thermodynamics; Thioredoxin; Spinacia oleracea
Año:2001
Volumen:40
Número:50
Página de inicio:15246
Página de fin:15256
DOI: http://dx.doi.org/10.1021/bi011186x
Título revista:Biochemistry
Título revista abreviado:Biochemistry
ISSN:00062960
CODEN:BICHA
CAS:thioredoxin, 52500-60-4; Amides; Protons; Solutions; Thioredoxin, 52500-60-4
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00062960_v40_n50_p15246_Neira

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Citas:

---------- APA ----------
Neira, J.L., González, C., Toiron, C., De Prat-Gay, G. & Rico, M. (2001) . Three-dimensional solution structure and stability of thioredoxin m from spinach. Biochemistry, 40(50), 15246-15256.
http://dx.doi.org/10.1021/bi011186x
---------- CHICAGO ----------
Neira, J.L., González, C., Toiron, C., De Prat-Gay, G., Rico, M. "Three-dimensional solution structure and stability of thioredoxin m from spinach" . Biochemistry 40, no. 50 (2001) : 15246-15256.
http://dx.doi.org/10.1021/bi011186x
---------- MLA ----------
Neira, J.L., González, C., Toiron, C., De Prat-Gay, G., Rico, M. "Three-dimensional solution structure and stability of thioredoxin m from spinach" . Biochemistry, vol. 40, no. 50, 2001, pp. 15246-15256.
http://dx.doi.org/10.1021/bi011186x
---------- VANCOUVER ----------
Neira, J.L., González, C., Toiron, C., De Prat-Gay, G., Rico, M. Three-dimensional solution structure and stability of thioredoxin m from spinach. Biochemistry. 2001;40(50):15246-15256.
http://dx.doi.org/10.1021/bi011186x