Structural and functional roles of a conserved proline residue in the alpha2 helix of Escherichia coli thioredoxin.

Frédéric de Lamotte-Guery 1 Christian Pruvost 2 Philippe Minard 3 Marc-André Delsuc 2 Myroslawa Miginiac-Maslow 1 Jean-Marie Schmitter 4 Mariana Stein 1 Paulette Decottignies 1
1 IBP - Institut de biotechnologie des plantes
2 CBS - Centre de Biochimie Structurale [Montpellier]
3 Laboratoire d'Enzymologie Physicochimique Moleculaire
4 BIOC - Laboratoire de Biochimie de l'Ecole polytechnique
Abstract : Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix. The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein. We have substituted Pro40 for Ala by using site-directed mutagenesis and expressed the mutant P40A in E.coli. The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations. Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3. The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agreement with the experimental results. The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol. However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation. Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.
Document type :
Journal articles
Complete list of metadatas
https://hal-polytechnique.archives-ouvertes.fr/hal-00788517
Contributor : Denis Roura <>
Submitted on : Tuesday, February 19, 2013 - 1:53:22 PM
Last modification on : Tuesday, May 28, 2019 - 11:02:04 AM

Links full text

openaccess

Identifiers

Collections

CNRS | X-BIOCH | X | PARISTECH | BS | UNIV-MONTPELLIER | X-DEP-BIO

Citation

Frédéric de Lamotte-Guery, Christian Pruvost, Philippe Minard, Marc-André Delsuc, Myroslawa Miginiac-Maslow, et al.. Structural and functional roles of a conserved proline residue in the alpha2 helix of Escherichia coli thioredoxin.. Protein Engineering, 1997, 10 (12), pp.1425-32. ⟨10.1093/protein/10.12.1425⟩. ⟨hal-00788517⟩

Export

BibTeX TEI DC DCterms EndNote

Share

Metrics

Record views

436

Contact

Informations

CCSD