Stabilization of human triosephosphate isomerase by improvement of the stability of individual alpha-helices in dimeric as well as monomeric forms of the protein.

Human triosephosphate isomerase (hTIM) is a dimeric enzyme of identical subunits, adopting the alpha/beta-barrel fold. In a previous work, a monomeric mutant of hTIM was engineered in which Met14 and Arg98, two interface residues, were changed to glutamine. Analysis of equilibrium denaturation of this monomeric mutant, named M14Q/R98Q, revealed that its conformational stability, 2.5kcal/mol, is low as compared to the stability of dimeric hTIM (19.3 kcal/mol). The fact that this value is also lower than the conformational stabilities usually found for monomeric proteins suggests that the hTIM monomers are thermodynamically unstable. In the present work, we attempted to stabilize the M14Q/R98Q mutant by introducing stabilizing mutations in alpha-helices of the protein. Five mutations were proposed, designed to increase alpha-helix propensity by introducing alanines at solvent-exposed sites (Q179A, K193A), to introduce favorable interactions with helix dipoles (Q179D, S105D), or to reduce the conformational entropy of unfolding by introducing proline residues at the "N-cap" position of alpha-helices (A215P). Three replacements (Q179D, K193A, and A215P) were found to increase the stability of the native dimeric hTIM and the monomeric M14Q/R98Q. These results suggest that the monomeric hTIM mutant can be stabilized to a considerable extent by following well-established rules for protein stabilization. A comparison of the stabilizing effect performed by the mutations on the dimeric hTIM and the monomeric M14Q/R98Q allowed us to reinforce a model of equilibrium denaturation proposed for both proteins.