Measuring protein conformational stability, especially on larger proteins, is one key to solving the protein folding problem. The traditional methods of measuring the conformational stability of a protein are solvent and thermal denaturation, but both require long extrapolations from the conditions required to promote unfolding to ambient conditions. In contrast, the conformational stability can be determined directly under ambient conditions from hydrogen-deuterium exchange rates measured using NMR, and can potentially be used with larger proteins that do not unfold by a two-state mechanism. For ribonuclease T1 and a variety of other proteins, we show that conformational stabilities determined by the traditional methods are in excellent agreement with those determined from hydrogen deuterium exchange rates if the effects of D2O and proline isomerization are taken into account. We also recommend that urea be used rather than guanidine hydrochloride when conformational stabilities are determined using the linear extrapolation method.

It is has proven difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. We will show that reversing the charge of 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 of 3.5 and a net charge of -6 at pH 7. The side chain of Asp 49 is hyperexposed, not hydrogen bonded, and 8 angstroms 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 (pK = 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. Our results show that protein stability can be increased by improving the Coulombic interactions among charged groups on the protein surface. We also show that a reverse hydrophobic effect is observed when nonpolar groups are substituted for Asp 49.

We will review the methods that look most promising for increasing the conformational stabilities of proteins based on our studies of the microbial ribonucleases.