Consolidation of the thioredoxin fold by peptide recognition: interaction between E. coli thioredoxin fragments 1-93 and 94-108.
Escherichia coli thioredoxin (TRX) catalyzes redox reactions via the reversible oxidation of the conserved active center WCGPC. TRX is a monomeric alpha/beta protein with a fold characterized by a central beta-sheet surrounded by alpha-helical elements. The interaction of the C-terminal alpha-helix (helix 5) of TRX against the remainder of the protein involves the close packing of hydrophobic surfaces, opening the possibility of studying a fine-tuned molecular recognition phenomenon. To evaluate the relevance of this interaction on the folding mechanism of TRX, we characterize TRX1-93, a truncated variant of TRX devoid of the last stretch of 15 amino acid residues that includes helix 5. TRX1-93 may possibly represent a molecular form where the folding process becomes interrupted, giving rise to a structure exhibiting the features of a molten globule state. This was assessed by circular dichroism, intrinsic fluorescence, binding of the probe ANS, size-exclusion chromatography, limited proteolysis, and calorimetry. Remarkably, fragment TRX1-93 interacts with peptide TRX94-108 (KD approximately 2-12 microM), bringing forth the restoration of native-like signatures and enzymic function. This represents a molecular event of reciprocal structure selection where both partners gain order, thus leading to long-range consequences on conformation. In this context, the binding of the C-terminal helix could signify a late event in the consolidation of the overall TRX fold.
Santos
Javier
J
Department of Biological Chemistry and Institute of Biochemistry and Biophysics (IQUIFIB), School of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956, C1113AAD Buenos Aires, Argentina.
Marino-Buslje
Cristina
C
Kleinman
Claudia
C
Ermácora
Mario R
MR
Delfino
José M
JM
D000595:Amino Acid Sequence; D002151:Calorimetry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D013879:Thioredoxins