A simple model for proteins with interacting domains. Applications to scanning calorimetry data

Abstract

A simple thermodynamic model is formulated for the purpose of interpreting scanning calorimetry data on proteins that have interacting domains. Interactions are quantified by inclusion of an interface free energy, .DELTA.GAB, in the thermodynamics of unfolding for multidomain proteins. The assumption is made that .DELTA.GAB goes to zero with the unfolding of either domain involved in pairwise interaction, so the interaction term appears to stabilize only the domain with the lower TM. Application of the model to calorimetric data leads to an estimate of -25,000 cal/mol for interactions between the regulatory and catalytic subunits of native aspartate transcarbamoylase and to a value of 0 for .DELTA.GAB between the transmembrane and cytoplasmic domains of band 3 of the human erythrocyte membrane. Estimates of changes in .DELTA.GAB are also obtained for mutant forms of yeast phosphoglycerate kinase that have been altered in the hinge region between amino-terminal and carboxy-terminal domains. The model is also applied to ligand binding to proteins having domains that communicate through pairwise interaction. It is shown that whenever the .DELTA.GAB term is ligand-dependent, then attachment of the ligand to the binding domain will be partially controlled by the other (regulatory) domain. This situation can sometimes be recognized and quantified when calorimetric scans are carried out at varying ligand concentrations. According to the model, the binding of MgATP to the carboxy-terminal domain of phosphoglycerate kinase is strongly stabilized (ca. 20% of the unitary free energy of binding) by participation of the amino-terminal domain, which acts to increase the binding constant 25-fold. A similar situation is found for binding of MgADP to creatine kinase, except the contribution from the regulatory domain is much smaller (ca. 5% of the unitary free energy of binding) and only increases the binding constant 2-fold.