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 identica l 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 equilibriu m denaturation of this monomeric mutant, named M14Q/R98Q, revealed tha t its conformational stability, 2.5 kcal/mol, is low as compared to th e stability of dimeric hTIM (19.3 kcal/mol). The fact that this value is also lower than the conformational stabilities usually found for mo nomeric proteins suggests that the hTIM monomers are thermodynamically unstable. In the present work, we attempted to stabilize the M14Q/R98 Q mutant by introducing stabilizing mutations in alpha-helices of the protein. Five mutations were proposed, designed to increase alpha-heli x 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 intro ducing proline residues at the ''N-cap'' position of alpha-helices (A2 15P). Three replacements (Q179D, K193A, and A215P) were found to incre ase the stability of the native dimeric hTIM and the monomeric M14Q/R9 8Q. These results suggest that the monomeric hTIM mutant can be stabil ized to a considerable extent by following well-established rules for protein stabilization. A comparison of the stabilizing effect performe d by the mutations on the dimeric hTIM and the monomeric M14Q/R98Q all owed us to reinforce a model of equilibrium denaturation proposed for both proteins.