Document Detail

Structural and functional roles of a conserved proline residue in the alpha2 helix of Escherichia coli thioredoxin.
MedLine Citation:
PMID:  9543004     Owner:  NLM     Status:  MEDLINE    
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.
F de Lamotte-Guéry; C Pruvost; P Minard; M A Delsuc; M Miginiac-Maslow; J M Schmitter; M Stein; P Decottignies
Related Documents :
22355224 - Study of the role of "gatekeeper" mutations v654a and t670i of c-kit kinase in the inte...
8360164 - Mutational study of streptomyces tyrosinase trans-activator melc1. melc1 is likely a ch...
22896704 - Microneme protein 5 regulates the activity of toxoplasma subtilisin 1 by mimicking a su...
22652334 - Assessment of drug-drug interactions caused by metabolism-dependent cytochrome p450 inh...
11004464 - The cytochrome b(6)f complex: structural studies and comparison with the bc(1) complex.
10430864 - A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen...
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Protein engineering     Volume:  10     ISSN:  0269-2139     ISO Abbreviation:  Protein Eng.     Publication Date:  1997 Dec 
Date Detail:
Created Date:  1998-05-15     Completed Date:  1998-05-15     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  8801484     Medline TA:  Protein Eng     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  1425-32     Citation Subset:  IM    
Institut de Biotechnologie des Plantes, ERS 569 CNRS, Université Paris XI, Orsay, France.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Amino Acid Sequence
Binding Sites
Chemistry, Physical
Chromatography, High Pressure Liquid
Conserved Sequence
Enzyme Activation
Escherichia coli / chemistry*
Malate Dehydrogenase / metabolism
Mass Spectrometry
Models, Molecular
Mutagenesis, Site-Directed
NADP / metabolism
Physicochemical Phenomena
Proline / chemistry*
Protein Folding
Protein Structure, Secondary*
Structure-Activity Relationship
Thioredoxins / chemistry*,  genetics,  metabolism*
Reg. No./Substance:
147-85-3/Proline; 52500-60-4/Thioredoxins; 53-59-8/NADP; EC Dehydrogenase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Previous Document:  A glutamine 67--> histidine mutation in homotetrameric R67 dihydrofolate reductase results in four m...
Next Document:  Spontaneous oligomerization of a staphylococcal alpha-hemolysin conformationally constrained by remo...