Figure 4.

A large Brønsted slope for H bonding in proteins is inferred from folding studies of Staphylococcal nuclease mutants. (A) Thermodynamic analysis depicting the effect of changing the strength of the H bond donor, the substituted tyrosine hydroxyl, on the stability of the protein. The folding equilibrium of a hypothetical non-H-bonded species (K_folding^{no HB}) is used to dissect the effects from H bonding. (B) Schematic depiction of the dependences of H bonding and folding equilibria on the pK_a value of substituted tyrosines. As the tyrosine hydroxyl becomes more acidic, the strengthening of its H bond to the enzymatic glutamate and to water stabilizes the folded and unfolded protein, respectively. The slope of the plot of log K_folding^obsd versus pK_a is the difference between the slopes of plots of log K_E^HB versus pK_a and K_water^HB versus pK_a. This follows from the thermodynamic relationship shown in A, which gives Δlog K_folding^obsd = Δlog K_E^HB − Δlog K_water^HB (i.e., the greater strengthening of the H bond on the protein results in a change in the observed stability of the protein. Note that Δlog K_folding^{no HB} = 0 by definition because the folding equilibrium between the non-H-bonded species does not depend on the strength of the H bond donor.