Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach
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- 作者：José ; L. Neira ; Carlos Gonzá ; lez ; Catherine Toiron ; Gonzalo de Prat-Gay ; and Manuel Rico
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：December 18, 2001
- 年：2001
- 卷：40
- 期：50
- 页码：15246 - 15256
- 全文大小：146K
- 年卷期：v.40,no.50(December 18, 2001)
- ISSN：1520-4995
文摘
Proton NMR spectral resonances of thioredoxin m from spinach have been assigned, and itssolution structure has been determined on the basis of 1156 nuclear Overhauser effect- (NOE-) deriveddistance 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 ± 0.1, whenthe 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 concludingthis work, a crystal structure of this protein was reported, in which thioredoxin m was found to crystallizeas noncovalent dimers. Although the solution and crystal structures are very similar, no evidence wasfound about the existence of dimers in solution, thus confirming that dimerization is not needed for theregulatory activity of thioredoxin m. The spinach thioredoxin m does not unfold by heat in the range25-85 
C, as revealed by thermal circular dichroic (CD) measurements. However, its unfolding freeenergy (9.1 ± 0.8 kcal mol-1, at pH 5.3 and 25 C) could be determined by extrapolating the free energyvalues obtained at different concentrations of guanidinium chloride (GdmCl). The folding-unfoldingprocess is two-state as indicated by the coincidence of the CD denaturation curves obtained at far andnear UV. The H/D exchange behavior of backbone amide protons was analyzed. The slowest-exchangingprotons, requiring a global-unfolding mechanism in order to exchange, are those from 
2, 3, and 4, thecentral strands of the -sheet, which constitute the main element of the core of the protein. The freeenergies obtained from exchange measurements of protons belonging to the 
-helices are lower thanthose derived from GdmCl denaturation studies, indicating that those protons exchange by local-unfoldingmechanisms.
C, as revealed by thermal circular dichroic (CD) measurements. However, its unfolding freeenergy (9.1 ± 0.8 kcal mol-1, at pH 5.3 and 25 C) could be determined by extrapolating the free energyvalues obtained at different concentrations of guanidinium chloride (GdmCl). The folding-unfoldingprocess is two-state as indicated by the coincidence of the CD denaturation curves obtained at far andnear UV. The H/D exchange behavior of backbone amide protons was analyzed. The slowest-exchangingprotons, requiring a global-unfolding mechanism in order to exchange, are those from 2, 3, and 4, thecentral strands of the -sheet, which constitute the main element of the core of the protein. The freeenergies obtained from exchange measurements of protons belonging to the -helices are lower thanthose derived from GdmCl denaturation studies, indicating that those protons exchange by local-unfoldingmechanisms.