Aim:

Monitoring equilibrium unfolding of protein using tryptophan fluorescence

Introduction:

Folding of a protein into its unique 3-dimensional structure is central for its function. The tertiary structure of a protein is determined by various intramolecular non-covalent interactions such as H-bonding, electrostatic interactions, and hydrophobic interactions. The conformational stability of the protein structure is an important parameter that defines and limits its utility. In this lecture, we shall see how the stability of a single domain globular protein is determined.

Folding/unfolding of small globular proteins closely approaches the two state folding/unfolding mechanisms:

(8.1)

The conformational stability of a small globular protein can be determined by calculating the equilibrium constant and the free energy, ΔG for the reaction shown in equation (8.1). The value of ΔG for the unfolding reaction shown in equation 8.1 in the absence of a denaturant is referred to as the conformational stability of a protein at a given temperature and is represented by ΔG (H₂O). Comparison of conformational stability of a protein with its variants allows determination of various forces and factors responsible for the protein's stability.

Methods of unfolding

The native structure of a protein is sensitive to its environment such as pH, temperature, ionic strength, cosolvents, and presence of denaturants. A change in any of these parameters can disrupt the non-covalent interactions thereby causing unfolding (denaturation) of protein. The conformational stability of a protein is most routinely determined by thermal denaturation or by denaturing the protein with urea or guanidinium chloride. Urea solutions have historically been used for determining the conformational stabilities of proteins. Although guanidinium chloride is a stronger denaturant and chemically more stable than urea, it is not preferred over urea because it is a salt and causes changes in the ionic strength of the solutions that could result in less reliable ΔG (H₂O). We shall, in this lecture, therefore be discussing the equilibrium unfolding of a protein using urea.