Calorimetric Analysis of Mutagenic Effects on Protein–Ligand Interactions

Abstract

Analysis of the effects of indirect T127→L mutagenesis on the thermodynamic changes in cooperativity of cAMP binding to cAMP receptor protein (CRP) has relied heavily on analysis of the structural changes induced in CRP by this mutation. This analysis results in the development of a structural model that best describes how the conformational changes induced by the T127→L mutation result in the observed thermodynamic changes in the cyclic nucleoside monophosphate (cNMP)–CRP binding interaction. The structural methods employed in this analysis of indirect mutagenesis effects included X-ray crystallography, small-angle neutron scattering (SANS), and circular dichroism (CD) measurements. Furthermore, it should be emphasized that this structural model offers an explanation based on the presently available structural and thermodynamic data about this system and could, thus, be changed as more experimental data about this intriguing system become available. Analysis of the effect of indirect mutagenesis on the thermodynamics of protein–ligand interactions involves development of a structural model to explain how mutations away from the binding site change the observed thermodynamics of the binding reaction: this is not as straight-forward as analysis of the effect of direct mutagenesis on the thermodynamics of protein–ligand interactions in terms of changes in the solvent-accessible surface areas at the binding site.