PMID: 9543004May 23, 1998Paper

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

Protein Engineering
F de Lamotte-GuéryP Decottignies

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/mo...Continue Reading

Citations

Feb 9, 2010·Antioxidants & Redox Signaling·Jean-Francois Collet, Joris Messens
Nov 19, 2003·FEBS Letters·Rosmarie FriemannHans Eklund
May 29, 2013·Chembiochem : a European Journal of Chemical Biology·Marina RubiniRudi Glockshuber
Mar 28, 2003·Biochimica Et Biophysica Acta·Kathryn M Taylor, Robert I Nicholson
Mar 21, 2007·Journal of Molecular Biology·Goedele RoosJoris Messens
Aug 5, 2000·Journal of Molecular Biology·J W SchymkowitzL S Itzhaki
Oct 28, 2018·Biological Chemistry·Sérgio F SousaMaria João Ramos
Apr 23, 2003·The Journal of Biological Chemistry·Valerie CollinMyroslawa Miginiac-Maslow
Jul 20, 2020·Journal of Structural Biology·María Daniela Torres-RodríguezFelipe Cruz-García
Nov 6, 2021·Biophysical Journal·Silvia NapolitanoAlvar D Gossert

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