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-) 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
![](/images/entities/deg.gif)
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
![](/images/entities/deg.gif)
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
![](/images/gifchars/beta2.gif)
2,
![](/images/gifchars/beta2.gif)
3, and
![](/images/gifchars/beta2.gif)
4, thecentral strands
of the
![](/images/gifchars/beta2.gif)
-sheet, which constitute the main element
of the core
of the protein. The freeenergies obtained from exchange measurements
of protons belonging to the
![](/images/gifchars/alpha.gif)
-helices are lower thanthose derived from GdmCl denaturation studies, indicating that those protons exchange by local-unfoldingmechanisms.