Thermodynamics of unfolding for kazal-type serine protease inhibitors: Entropic stabilization of ovomucoid first domain by glycosylation

A synthetic gene for chicken ovomucoid first domain (OMCHI1) has been overexpressed in Escherichia coli. The resulting recombinant protein, rOMCHI1, is expressed and correctly folded without the use of fusion proteins or export secretion signal peptides incorporated into the gene. The thermostability of rOMCHI1 has been compared to that of the naturally occurring glycosylated OMCHII (gOMCHI1). The results of differential scanning calorimetry (DSC) studies show that the heat capacity change for unfolding, ΔC(p), for both rOMCHI1 and gOMCHI1 is approximately 600 cal/(mol·K). At any given pH, however, the presence of N-linked carbohydrate increases the T(m) for thermal unfolding of gOMCHI1 over rOMCHI1 by 2-4 °C, without changing the enthalpy of unfolding, ΔH°(m). This suggests that the increased thermal stability of gOMCHI1 is entropic. Comparison of the unfolding thermodynamics of rOMCHI1 with those of turkey ovomucoid third domain (OMTKY3), which is 36% identical to rOMCHI1, reveals similar ΔC(p) values for both proteins, about 600 cal/(mol·K), but a reduction in ΔH°(m) of about 5 kcal/mol for rOMCHI1 at all temperatures. Decreases in ΔH°(m) for rOMCHI1 versus OMTKY3 may be explained by an overall less ordered native state in rOMCHI1. In the absence of a native structure for OMCHI1, the change in accessible surface area upon unfolding, ΔASA, was calculated using unfolding parameters and structural energetic relationships [Murphy and Freire (1992) Adv. Protein Chem. 43, 313361; Murphy et al. (1993), Proteins: Struct., Funct., Genet. 15, 113-120]. These calculations suggest that the larger protein rOMCHI1 (M(r) 7500) exposes less surface area than OMTKY3 (M(r) 6100) upon thermal denaturation. Overall, structural energetic relationships may provide a useful framework for interpretation and comparison of thermodynamic data for structurally homologous proteins.