Increasing Enzyme Stability

Abstract

Enzymes are active only when their polypeptide chain is folded into a unique globular conformation with a functional active site. In most naturally occurring enzymes, this globular conformation is only 5 to 10 kcal/mole more stable than unfolded, biologically inactive conformations (1). The conformational stability of a protein is defined as the free energy change for the reaction: $$ \textup{folded}\;\textup{(native)}\; \;\textup{unfolded\;(denatured)} $$ (1) under ambient conditions, such as water at 25°C. The most convenient methods of estimating the conformational stability of a protein are urea (or guanidine hydrochloride (GdnHCl)) unfolding curves and thermal unfolding curves. Estimates of the conformational stability based on urea unfolding curves are designated ΔG(H2O), and estimates from thermal unfolding curves are designated ΔG(25°C). In the first part of this article, we describe how to measure ΔG(H2O) and ΔG(25°C).