Engineering the independent folding of the subtilisin BPN' prodomain: Analysis of two-state folding versus protein stability

Sergei Ruvinov, Lan Wang, Biao Ruan, Orna Almog, Gary L. Gilliland, Edward Eisenstein, Philip N. Bryan

Research output: Contribution to journalArticlepeer-review

41 Scopus citations

Abstract

In complex with subtilisin BPN', the 77 amino acid prodomain folds into a stable compact structure comprising a four-stranded antiparallel β-sheet and two three-turn α-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 ΔG(unfolding) 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 (ΔG = 0). Evidence for its two-state folding is based on the observed additivity of individual mutations to the overall ΔG(unfolding) and the conformity of ΔG(unfolding) 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.

Original languageEnglish
Pages (from-to)10414-10421
Number of pages8
JournalBiochemistry
Volume36
Issue number34
DOIs
StatePublished - 26 Aug 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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