Increasing protein stability by altering long-range coulombic interactions

It is difficult to increase protein stability by adding hydrogen bonds or b urying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of incr easing stability. Ribonuclease T1 is an acidic protein with a pI approximat e to 3.5 and a net charge of approximate to -6 at pH 7. The side chain of A sp49 is hyperexposed, not hydrogen bonded, and 8 Angstrom from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild- type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild-type at pH 6, where the histidine 49 side ch ain (pK(a) = 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mo l more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among c harged groups on the protein surface. In addition, the stability of RNase T I decreases as more hydrophobic aromatic residues are substituted for Ala49 , indicating a reverse hydrophobic effect.