Ernesto Freire

Many proteins are intrinsically disordered or contain one or more disordered domains. These domains can participate in binding interactions with other proteins or small ligands. Binding to intrinsically disordered protein domains requires the folding or structuring of those regions such that they can establish well-defined stoichiometric interactions. Since, in such a situation binding is coupled to folding, the energetics of those two events is reflected in the measured binding thermodynamics. In this protocol, we illustrate the thermodynamic differences between binding coupled to folding and binding independent of folding for the same protein. As an example, we use the HIV-1 envelope glycoprotein gp120 that contains structured as well as disordered domains. In the experiments presented, the binding of gp120 to molecules that bind to disordered regions and trigger structuring (CD4 or MAb 17b) and to molecules that bind to structured regions and do not induce conformational structuring (MAb b12) is discussed.

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (∼100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day⁻¹. Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.