Increasing protein stability: Importance of ΔC_p and the denatured state

Abstract

Increasing the conformational stability of proteins is an important goal for both basic research and industrial applications. In vitro selection has been used successfully to increase protein stability, but more often site-directed mutagenesis is used to optimize the various forces that contribute to protein stability. In previous studies, we showed that improving electrostatic interactions on the protein surface and improving the b-turn sequences were good general strategies for increasing protein stability, and used them to increase the stability of RNase Sa. By incorporating seven of these mutations in RNase Sa, we increased the stability by 5.3 kcal/mol. Adding one more mutation, D79F, gave a total increase in stability of 7.7 kcal/mol, and a melting temperature 28°C higher than the wild-type enzyme. Surprisingly, the D79F mutation lowers the change in heat capacity for folding, DC p , by 0.6 kcal/mol/K. This suggests that this mutation stabilizes structure in the denatured state ensemble. We made other mutants that give some insight into the structure present in the denatured state. Finally, the thermodynamics of folding of these stabilized variants of RNase Sa are compared with those observed for proteins from thermophiles.

Contribution of Hydrophobic Interactions to Protein Stability

“…40–45 To study the contribution of the hydrophobic effect to the stability of RNase Sa, we prepared 5 Ile to Val and 5 Leu to Ala mutants. Three thermal denaturation curves were determined for each mutant and the average results are summarized in Table 4.…”

Residual structure in unfolded proteins

“…Recently, measurements of Δ C p coupled to site-directed mutagenesis have been used to define loci of residual structure in the DSE. For RNase Sa, a D79F mutation decreases Δ C p from 1.68 to 1.07 kcal mol −1 K −1 [33]. Second site variants indicate that F79, I92 and Y80 stabilize a nativelike hydrophobic cluster in the DSE.…”

“…All 3 aromatic residues stabilized the loop by 0.4 to 0.5 kcal/mol in 3 M GdnHCl, whereas leucine had a negligible effect on loop stability. While the changes in loop stability are small in magnitude, the fact that single aromatic or aliphatic to alanine mutations are adequate to break up hydrophobic clusters in the DSE of RNase Sa [33] and RNase H1 [34] suggests that interactions of modest magnitude are sufficient to change the structural bias of the DSE. For all three alanine to aromatic substitutions, the stabilization of the His-heme loop was due primarily to a decrease in the rate constant for His-heme loop breakage.…”

Propensities of Aromatic Amino Acids versus Leucine and Proline to Induce Residual Structure in the Denatured-State Ensemble of Iso-1-cytochrome c

“…23-28 Mutation of charged residues to hydrophobic residues has been shown to decrease the heat capacity change, Δ C p , for thermal unfolding consistent with formation of hydrophobic clusters in the DSE. 29,30 Simulation and experiment on the drkN SH3 domain, barnase and hen egg white lysozyme have led to the conclusion that aromatic residues, in particular, promote hydrophobic clusters in the DSE 10,11,14,31-37. However, simulation and experiment on chymotrypsin inhibitor 2 and α-lactalbumin show that larger aliphatic side chains are also sufficient to nucleate hydrophobic clusters in the DSE.…”