The two most common methods of measuring the conformational stability of a protein are differential scanning calorimetry and an analysis of solvent denaturation curves by using the linear extrapolation method. In this article, we trace the history of the linear extrapolation method, review how the method is used to measure protein stability, and then discuss some of the other important uses. Proteins 2000;Suppl 4:1–7. © 2000 Wiley‐Liss, Inc.

The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI ‫ס‬ 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI ‫ס‬ 6.4 and a 5K variant (3K + D25K, E74K) with a pI ‫ס‬ 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.

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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 Ϸ 3.5 and a net charge of ϷϪ6 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 kcal0mol greater than wild-type at pH 7 and 0.4 kcal0mol less at pH 2.5. The stability of Asp49His is 1.1 kcal0mol greater than wild-type at pH 6, where the histidine 49 side chain~pK a ϭ 7.2! is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal0mol and Glu74Lys is 1.1 kcal0mol 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.

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