Engineering the Independent Folding of the Subtilisin BPN‘ Prodomain:  Analysis of Two-State Folding versus Protein Stability

Abstract

In complex with subtilisin BPN', the 77 amino acid prodomain folds into a stable compact structure comprising a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated from subtilisin, the prodomain is 97% unfolded even under optimal folding conditions. Traditionally, to study stable proteins, denaturing cosolvents or temperatures are used to shift the equilibrium from folded to unfolded. Here we manipulate the folding equilibrium of the unstable prodomain by introducing stabilizing mutations generated by design. By sequentially introducing three stabilizing mutations into the prodomain we are able to shift the equilibrium for independent folding from 97% unfolded to 65% folded. Spectroscopic and thermodynamic analysis of the folding reaction was carried out to assess the effect of stability on two-state behavior and the denatured state. The denatured states of single and combination mutants are not discernably different in spite of a range of DeltaGunfolding from -2.1 to 0.4 kcal/mol. Conclusions about the nature of the denatured state of the prodomain are based on CD spectral data and calorimetric data. Two state folding is observed for a combination mutant of marginal stability (DeltaG = 0). Evidence for its two-state folding is based on the observed additivity of individual mutations to the overall DeltaGunfolding and the conformity of DeltaGunfolding vs T to two-state assumptions as embodied in the Gibbs-Helmholz equation. We believe our success in stabilizing the two-state folding reaction of the prodomain originates from the selection of mutations with improved ability to fold subtilisin rather than selection for increase in secondary structure content. The fact that a small number of mutations can stabilize the independent folding of the prodomain implies that most of the folding information already exists in the wild-type amino acid sequence in spite of the fact that the unfolded state predominates.

Stabilizing the subtilisin BPN' pro‐domain by phage display selection: How restrictive is the amino acid code for maximum protein stability?

“…This thermodynamic linkage between stability and binding has been demonstrated previously using a series of designed mutations, which increase the stability of the pro-domain (Ruvinov et al, 1997).…”

“…Based on the equilibrium sedimentation data, pro-RI is 99% monomeric under these conditions at pH 5.0. The equilibrium constant for folding in 100 mM NaOAc, pH 5.0 is the same as the intrinsic equilibrium constant for folding (independent of dimerization) in 100 mM KPI, pH 7.0 (Ruvinov et al, 1997). The CD spectrum of pro-wt is typical of a largely random coil structure with a minimum ellipticity at 198 nM (Fig.…”

“…The pro-RI mutant illustrates that optimizing one set of local interactions results in a stable unique fold. We have shown previously that introduction of three mutations in different regions of the pro-domain stabilizes the native fold by 2.5 kcal/mol (Ruvinov et al, 1997). An overemphasis on the binary pattern may result in designed proteins in which secondary structural elements have high independent stability at the expense of the stability of specific tertiary interactions (Cordes et al, 1996).…”

“…Analysis of the stability was carried out in 100 mM NaOAc, pH 5.0 at [PI = 50 pM. Previous studies have shown that stabilized pro-domains have a tendency to dimerize at pH 7.0 (Ruvinov et al, 1997). Based on the equilibrium sedimentation data, pro-RI is 99% monomeric under these conditions at pH 5.0.…”

“…If mutations are introduced in regions of the pro-domain that do not directly contact subtilisin, their effects on binding to subtilisin will be linked to whether or not they stabilize the native conformation. Therefore, mutations that stabilize independent folding of the pro-domain will increase its binding affinity to subtilisin (Ruvinov et al, 1997). Two libraries of mutant pro-domains were constructed and analyzed.…”