LINKAGE OF THIOREDOXIN STABILITY TO TITRATION OF IONIZABLE GROUPS WITH PERTURBED PKA

The highly conserved, buried, Asp 26 in Escherichia coli thioredoxin has a pK(a) = 7.5, and its titration is associated with a sizable destabilization of the protein [Langsetmo, K., Fuchs, J., & Woodward, C. (1991) Biochemistry (preceding paper in this issue)]. A fit of the experimental pH dependence of thioredoxin stability to a theoretical expression for the pH/stability relation in proteins agrees closely with a pK(a) value of 7.5 for Asp 26. The agreement between the experimental and theoretical changes in protein stability due to substitution of Asp 26 by alanine is also good. The local structure in the vicinity of Asp 26 in the low-pH crystal structure (with uncharged Asp 26) is hydrophobic, indicating that the aspartate would be highly destabilized. In theoretical calculations, the desolvation penalty for deprotonating Asp 26 in this environment is similar to the total protein folding energy. As a consequence, the Asp 26 pK(a) would be much greater than 7.5, and/or the protein might not fold. This suggests that a compensating process partially stabilizes the Asp 26 carboxyl group when it is charged. A simple model for this is proposed, whereby the Lys 57 side chain rotates to form a salt bridge with Asp 26 when it is deprotonated.