Stabilization of human pancreatic ribonuclease through mutation at its N-terminal edge.

Enzyme stability can be an important parameter in the design of recombinant toxins because unstable proteins are often degraded before they can reach their cellular target. There is great interest in the design of human pancreatic ribonuclease variants that could be cytotoxic against tumoral cells. To this end, some residues in the protein need to be substituted, but this may result in a loss of stability. Previous papers have reported the production of N- and C-terminal human pancreatic ribonuclease variants with increased thermal stability. Here, we investigated the contribution of the different amino acid changes at the N-terminus of the protein to its thermostability increase. We show that this increase correlates with the helical propensity of the first alpha-helix of the protein. On the other hand, deletion of the four last residues of the protein does not affect its thermal stability. These results set the basis for the design of a human pancreatic ribonuclease template on which amino acid substitutions can be made that could render the enzyme cytotoxic, without an important loss in its stability.