It is difficult to increase protein stability by
adding hydrogen bonds or burying 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 increasing stability. Ribonuclease T1 is an acidic protein
with a pI ≈ 3.5 and a net charge of ≈−6 at
pH 7. The side chain of Asp49 is hyperexposed, not hydrogen
bonded, and 8 Å 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 chain (pKa
= 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/mol more stable than the wild-type
enzyme. These results suggest that protein stability can
be increased by improving the coulombic interactions among
charged groups on the protein surface. In addition, the
stability of RNase T1 decreases as more hydrophobic aromatic
residues are substituted for Ala49, indicating a reverse
hydrophobic effect.