STRUCTURAL AND FUNCTIONAL ROLES OF A CONSERVED PROLINE RESIDUE IN THEALPHA-2 HELIX OF ESCHERICHIA-COLI THIOREDOXIN

Proline 40 in Escherichia coli thioredoxin is located close to the red ox active site (Cys32-Cys35) within the alpha 2 helix. The conservatio n of this residue among most of the thioredoxins suggests that it coul d play an important role in the structure and/or function of this prot ein. We have substituted Pro40 for Ala by using site-directed mutagene sis and expressed the mutant P40A in E.coli. The effects of the mutati on on the biophysical and biological properties of thioredoxin have be en 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 cle ft located between helices alpha 2 and alpha 3. The solvation free ene rgy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agre ement with the experimental results. The mutant P40A exhibited chromat ographic behavior and fluorescence properties very different from thos e of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A sho wed that the mutant was globally destabilized by 2.9 kcal/mol. However , the effect of the mutation on the transition curve was highly unusua l as the midpoint of the unfolding transition increased, indicating th at some local structures were actually stabilized by the mutation. Des pite these structural modifications, neither the ability of the protei n to reduce a chloroplastic enzyme nor its reactivity with the bacteri al reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase und er air, which suggests that the mutant was less rapidly re-oxidized th an WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required f or the stability of the protein.