Urea and Guanidine Hydrochloride Denaturation Curves

Abstract

Proteins are synthesized as a linear chain of amino acids. In order to become biologically active, a protein must fold and adopt one out of an enormous number of possible conformations. This conformation, which we will refer to as the native state, exists in solution as a very compact, highly ordered structure. This native state structure results from a delicate balance between large and opposing forces. In order to form the native state, the forces that favor the unfolded state (mainly conformational entropy) must be overcome by the covalent and noncovalent interactions favoring the folded protein (see Chapter 1). Under physiological conditions the native (folded) and the denatured (unfolded) states of a protein are in equilibrium. The free energy change, △G, for the equilibrium reaction

(1)

is referred to as the conformational stability of a protein. The determinants of native state stability in aqueous solutions are the amino acid sequence of the protein as well as the variable conditions of pH, temperature, and the concentration of salts and ligands (1, 2). Although the native conformation is essential for activity, the conformational stability is remarkably low. The native state of most naturally occurring proteins is only about 5–15 kcal/mol more stable than its unfolded conformations (3).